CN101149372A - Immunofluorescence label reagent kit - Google Patents
Immunofluorescence label reagent kit Download PDFInfo
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- CN101149372A CN101149372A CNA2007100141955A CN200710014195A CN101149372A CN 101149372 A CN101149372 A CN 101149372A CN A2007100141955 A CNA2007100141955 A CN A2007100141955A CN 200710014195 A CN200710014195 A CN 200710014195A CN 101149372 A CN101149372 A CN 101149372A
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Abstract
This invention discloses to a kind of immunofluorescence mark reagent box. It is a reagent box applied for the aspects of immunity mark which uses the green fluorescence protein (GFP) as the indicator, the staphylococcus protein A (SPA) as the antibody. It also discloses the preparation method of the reagent. This invention can applied wide range in the field such as the immunity fluorescence mark, immunity histochemistry, biochemistry analysis, antibody test, protein chip and so on. It can replace the exist product efficiently, its operation is more simple, stability, delicacy and reliability, it is especially the same with the high flux analysis which analyze by the fluorescence analysis instrument.
Description
Technical field
The present invention is is indicator with the green fluorescent protein, be antiantibody with the staphylococcal protein A, be applied to the kit of immune labeled aspect, it relates to immunofluorescence label, immunohistochemistry, biochemical analysis, antibody test, protein fusion, green fluorescent protein, albumin A, genetic engineering etc., belongs to association areas such as immunoassay and biotechnology applications.
Background technology
Since the label immunoassay technology occurred, it was widely used in medical science and biological study.Its principle is exactly that the specificity of antigen-antibody reaction and susceptibility are combined with the accuracy of micro-spike, with colour developing or fluorescent material labelled antibody, tissue or intracellular antigenic substance is positioned detection.
In immunoassays, be essential with enzyme or fluorescent material labelled antibody/antigen.Traditional labeling method is that indicant is coupled on the antibody/antigen molecule (as enzyme is attached on the protein molecular by having crosslinking chemical difunctional or the multifunctional chemical group by chemical crosslink technique.But this method has its shortcoming: a large amount of antibody or antigen molecule, enzyme are participated in coupling as reactant; Need purification step just can obtain antibody-enzyme couplings; Crosslinked back enzymatic activity reduces often; And the sterically hindered interference that causes because of random incorporation in the mensuration process influences the sensitivity of immunoassays.In addition, the photobleaching phenomenon easily takes place in the conventional fluorescent dyestuff under the fluorescent microscope excitation light irradiation, and traditional Antibody Preparation and labeling method easily reduce antibody titer and produce unspecific staining.Therefore develop new label and labeling method is highly significant.
Green fluorescent protein (GFP) is the protein from a kind of function uniqueness of oceanic invertebrates such as luminescent jellyfish, and it is subjected to ultraviolet or when blue-light excited, can launches green fluorescence.Since GFP gene in 1994 was cloned success, it had huge application potential as the biomarker molecule, and main because it has following characteristic: (1) kind is dependence not, all can be expressed as activated GFP in protokaryon, the eukaryotic; (2) generation of fluorescence does not need reaction substrate and accessory factor; (3) less relatively molecule and monomeric protein make it to be easy to merge; (4) the alternative fluorescence that can make it to produce different colours of some specific amino acids in the GFP peptide chain can be adapted to the different needs of studying; (5) GFP can be used as a kind of non-intruding method (need not the cell permeation, toxic effects) and detects gene expression in vivo or protein labeling; (6) its conformation is stable, no photobleaching, fluorescence intensity height; (7) a series of N end or C with GFP are held in the property research of fusion, proved that fusion has the photoluminescent property of GFP and the biological function of ligandin matter.
Staphylococcal protein A (SPA) is that the isolated a kind of molecular weight of aureus cell wall is 42000 single polypeptide chain.SPA has many biologic activity such as activating complement, mitogenesis, inhibition is engulfed etc.Wherein SPA can with the Fc fragment non-specific binding of IgG in people and the multiple mammalian blood serum, and this combination does not influence the immunocompetence that the Fab fragment combines with antigentic specificity, utilize this characteristic, set up many sensitivities, special, quick, easy experimental technique in conjunction with technology such as radiation, fluorescence, enzyme connection and immuno-electron microscopes, be used for the research of medical diagnosis on disease and others.SPA is except that being used for immunoglobulin (Ig) and Purification of Monoclonal Antibodies at present, with biotin (biotin), peroxidase (peroxidase), fluorescein isothiocynate (FITC), collaurum labelled protein SPA such as (colloidal gold), in SABC, immuno-electron microscope, Western Blotting and ELISA, obtain widely applying, be called as " extensive two is anti-".
CN1731181 discloses a kind of immunofluorescence detection agent, is made up of the fusion (ProA-EGFP) of enhanced green fluorescence protein and staphylococcal protein A.Also disclose the preparation method of this reagent simultaneously, promptly made up green fluorescent protein (GFP) and staphylococcal protein A (ProA) integrative gene expression vector, expressed the ProA-EGFP fusion with Escherichia coli E.coli DH5 α bacterial strain; Separation and purification ProA-EGFP fusion.Immunofluorescence detection agent of the present invention has widely in preparation immunology diagnosis or detectable to be used.This patented claim at be the preparation of immunofluorescence reagent.The application at be the concrete application of this reagent.
Summary of the invention
At the deficiencies in the prior art, the invention provides a kind of immunofluorescence label reagent kit and preparation method thereof.
Immunofluorescence label reagent kit of the present invention is a kind of immunofluorescence label reagent kit that uses green fluorescent protein as fluorescence indicator.
Immunofluorescence label reagent kit of the present invention, composed as follows:
The fusion SPA-EGFP that enhanced green fluorescence protein (EGFP) and staphylococcal protein A (SPA) constitute, sealer, elution buffer.
Described sealer is 0.5% bovine serum albumin(BSA).
Described elution buffer is the 10mM PBS that contains 0.5% polysorbas20, pH7.4.
The preparation method of immunofluorescence label reagent kit of the present invention, the antigen-4 fusion protein gene spa-egfp of structure enhanced green fluorescence protein and staphylococcal protein A, with Pichia anomala expression SPA-EGFP fusion, separation and purification SPA-EGFP fusion; The SPA-EGFP fusion of separation and purification constitutes kit jointly as fluorescence indicator and sealer, elution buffer.
Described SPA-EGFP fusion is a yeast secreted expression, the effect expression vector is plasmid pPIC9K, primer is by design routinely, SPA-EGFP antigen-4 fusion protein gene spa-egfp inserts α-factor signal peptide downstream of plasmid pPIC9K, expressing with host cell is Pichia pastoris GS115, electricity transforms Pichia pastoris and the high copy of screening recon, makes up reorganization spa-egfp fusion Pichia yeast engineering.
Above-mentioned Pichia yeast engineering is fermented, and method is as follows:
Use the BMMY nutrient culture media, fermentation condition is: 30 ℃, and methanol concentration 2%, pH of buffer value 7.0 is added 1% acid hydrolysis casein (Casamino acids) simultaneously.The 300rpm shaking table is cultivated 80～84h, adds methyl alcohol to final concentration 2% in per 20～24 hours.
Above-mentioned BMMY culture medium prescription: 1% yeast extract, 2% polyprotein peptone, 100mM phosphate buffer, 1.34% no amino yeast nitrogen, 4 * 10
-5The % biotin, 2% methyl alcohol, 1% acid hydrolysis casein (Casaminoacids).
Above-mentioned Pichia yeast engineering fermentation liquor is centrifugal, to get supernatant and cross 0.45 micron filter membrane, last HisTrap HP post purifying after the desalination of HiTrap Desalting post, obtains purity greater than 99% SPA-EGFP.
The SPA-EGFP fusion freeze-drying of purifying is concentrated, and the SPA-EGFP fusion that is obtained is settled to finite concentration with dilution buffer liquid, constitutes kit jointly as fluorescence indicator and sealer, elution buffer.
Described SPA-EGFP fusion can be secreted to nutrient culture media after expressing in yeast effectively, and expression product possesses the fluorescence activity of the IgG of SPA in conjunction with activity and EGFP simultaneously.
Immunofluorescence label reagent kit of the present invention can be used for preparing the detection kit.
Immunofluorescence label reagent kit of the present invention can be used for preparing detection kit.
Immunofluorescence label reagent kit of the present invention can be used for the research of aspects such as amynologic label, the detection of cellular immunity group and protein chip.
The present invention is by the DNA recombinant technique, with enhanced green fluorescence protein (EGFP) gene and staphylococcal protein A (SPA) gene fusion, with the fusion transfection in Pichia pastoris GS115, and make it efficiently express SPA-EGFP, expression product is secreted to nutrient culture media, obtained highly purified SPA-EGFP fusion behind the purifying.Utilize this fusion to replace indicator system in traditional immune labeled kit (enzyme connection colour developing/fluorescent reagent) and antiantibody (also can be described as two resists), cooperation constitutes novel immune labeled kit with sealer, the elution buffer solution of special use.
Green fluorescent protein immunofluorescence label reagent kit of the present invention, be indicator, be antiantibody with enhanced green fluorescence protein (EGFP) with staphylococcal protein A (SPA), can be widely used in fields such as immunofluorescence label, immunohistochemistry, biochemical analysis, antibody test, protein-chip, can effectively replace existing product, and operate easier, stability, sensitivity and better reliability are particularly useful for the high throughput analysis that adopts fluorescence analyzer to carry out.
Embodiment
Embodiment 1: immunofluorescence label reagent kit
Composed as follows: the fusion SPA-EGFP that enhanced green fluorescence protein and staphylococcal protein A constitute, the bovine serum albumin(BSA) of sealer 0.5%, elution buffer contain the 10mMPBS of 0.5%Tween 20, pH7.4.
Embodiment 2: the preparation of immunofluorescence label reagent kit
1, fusion SPA-EGFP construction expression method is referring to CN1731181.
(plasmid is a dna sequence dna to SPA-EGFP antigen-4 fusion protein gene spa-egfp, have only gene just can insert in the plasmid) α-factor signal peptide downstream of inserting plasmid pPIC9K, expressing with host cell is Pichia pastoris GS115, electricity transforms Pichia pastoris and the high copy of screening recon, makes up reorganization SPA-EGFP fusion Pichia yeast engineering.Above-mentioned Pichia yeast engineering is fermented, use the BMMY nutrient culture media, fermentation condition is: 30 ℃, and methanol concentration 2%, pH of buffer value 7.0 is added 1%Casaminoacids simultaneously.The 300rpm shaking table is cultivated 80～84h, adds methyl alcohol to final concentration 2% in per 20～24 hours.The Pichia yeast engineering fermentation liquor is centrifugal, to get supernatant and cross 0.45 micron filter membrane, last HisTrap HP post purifying after the desalination of HiTrap Desalting post, obtains purity greater than 99% SPA-EGFP.The SPA-EGFP fusion freeze-drying of purifying is concentrated, standby.
2, the bovine serum albumin(BSA) of employing 0.5% is as sealer.
3, adopt contain 0.5%Tween 20 50mM PBS as elution buffer, its pH7.4.
4, the purifying SPA-EGFP fusion that is obtained is settled to 1: 100 (quality percent by volume) with dilution buffer liquid, constitutes kit jointly as fluorescence indicator and sealer, elution buffer.
Above-mentioned dilution buffer liquid specifically is 20mM PBS, and it is pH8.0.The dilution buffer liquid of the following examples 3-4 is identical with present embodiment.
Embodiment 3: the using method of immunofluorescence label reagent kit (direct method)
Dilution: as required, with dilution buffer liquid with antigen diluent to finite concentration.
Fixing: as will to dilute good antigen and drop on the solid phase carrier, and cover no antigen zone, and avoid the non-specific adsorption of fluorescence indicator with sealer.
Mark: fluorescence indicator is diluted to the experiment desired concn, with sample mix, incubation, to make it and antibodies.
Application of sample: drip the good sample of mark to the solid phase carrier that is fixed with antigen, incubation is so that the antigen-antibody combination.
Clean: wash solid phase carrier three times with elution buffer, remove not in conjunction with sample and fluorescence indicator.
The result observes: carry out qualitative/quantitative observation under the fluorescent microscope.
Embodiment 4: the using method of immunofluorescence label reagent kit (indirect method)
Dilution: as required, with dilution buffer liquid with antigen diluent to finite concentration.
Fixing: as will to dilute good antigen and drop on the solid phase carrier, and cover no antigen zone, and avoid the non-specific adsorption of fluorescence indicator with sealer.
Sample preparation: use dilution buffer liquid with diluted sample to finite concentration.
Application of sample: the sample drop of handling well is added on the solid phase carrier that is fixed with antigen, and incubation is so that the antigen-antibody combination.
Mark: with fluorescence indicator drop to antigen-antibody in conjunction with after solid phase carrier on, to make it and antibodies.
Clean: wash solid phase carrier three times with elution buffer, remove not in conjunction with sample and fluorescence indicator.
The result observes: carry out qualitative/quantitative observation under the fluorescent microscope.
Claims (7)
1. immunofluorescence label reagent kit that uses green fluorescent protein as fluorescence indicator.
2. immunofluorescence label reagent kit as claimed in claim 1 is characterized in that being made up of following reagent: the SPA-EGFP fusion that enhanced green fluorescence protein (EGFP) and staphylococcal protein A (ProA) constitute, sealer, elution buffer; Described sealer is 0.5% bovine serum albumin(BSA), and described elution buffer is the 10mM PBS that contains 0.5% polysorbas20, pH7.4.
3. immunofluorescence label reagent kit as claimed in claim 2 is characterized in that the SPA-EGFP fusion is a yeast secreted expression, and expressing with host cell is Pichia pastoris GS115.
4. immunofluorescence label reagent kit as claimed in claim 2 is characterized in that the spa-egfp fusion is inserted among the Yeast expression carrier pPIC9K, and this expression vector is transformed Pichia pastoris GS115, is built into SPA-EGFP fusion Pichia yeast engineering.
5. immunofluorescence label reagent kit as claimed in claim 3 is characterized in that secreting effectively to nutrient culture media after the SPA-EGFP fusion is expressed in yeast, and expression product possesses the fluorescence activity of the IgG of SPA in conjunction with activity and EGFP simultaneously.
6. the application of the described immunofluorescence label reagent kit of claim 1 in the preparation detectable.
7. the application of the described immunofluorescence label reagent kit of claim 1 on aspects such as preparation amynologic label, the detection of cellular immunity group and protein chip.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100141955A CN101149372A (en) | 2007-04-16 | 2007-04-16 | Immunofluorescence label reagent kit |
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| CNA2007100141955A CN101149372A (en) | 2007-04-16 | 2007-04-16 | Immunofluorescence label reagent kit |
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| CN101149372A true CN101149372A (en) | 2008-03-26 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101915836A (en) * | 2010-07-23 | 2010-12-15 | 中国检验检疫科学研究院 | Protein suspension chip for detecting dengue antibody in serum sample and preparation method and using method thereof |
| CN101936988A (en) * | 2010-07-23 | 2011-01-05 | 中国检验检疫科学研究院 | Protein suspension chip for detecting west nile antibody in serum sample and preparation method and using method thereof |
| CN101936989A (en) * | 2010-07-23 | 2011-01-05 | 中国检验检疫科学研究院 | Protein suspension chip for synchronously detecting various antibodies in serum sample and preparation method and using method thereof |
| CN102313813A (en) * | 2008-05-20 | 2012-01-11 | 北京莱尔生物医药科技有限公司 | Integration method for enriching and detecting rare cells from biological fluid samples |
| CN103694358A (en) * | 2013-12-26 | 2014-04-02 | 潍坊医学院 | Locus specificity biotin labeled recombinant IgG (Intravenous Gamma Globulin) affine peptide and application thereof in IgG antibody three-dimensional oriented fixation |
| CN107085109A (en) * | 2017-05-15 | 2017-08-22 | 山东大学深圳研究院 | Application of the high positive charge green fluorescent protein in hepatocarcinoma early diagnosis kit is prepared |
| CN117092344A (en) * | 2023-07-19 | 2023-11-21 | 武汉睿奇生物工程有限公司 | Kit for detecting staphylococcus aureus protein A and application thereof |
-
2007
- 2007-04-16 CN CNA2007100141955A patent/CN101149372A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102313813A (en) * | 2008-05-20 | 2012-01-11 | 北京莱尔生物医药科技有限公司 | Integration method for enriching and detecting rare cells from biological fluid samples |
| CN102313813B (en) * | 2008-05-20 | 2014-01-15 | 北京莱尔生物医药科技有限公司 | Integration method for enriching and detecting rare cells from biological fluid samples |
| CN101915836A (en) * | 2010-07-23 | 2010-12-15 | 中国检验检疫科学研究院 | Protein suspension chip for detecting dengue antibody in serum sample and preparation method and using method thereof |
| CN101936988A (en) * | 2010-07-23 | 2011-01-05 | 中国检验检疫科学研究院 | Protein suspension chip for detecting west nile antibody in serum sample and preparation method and using method thereof |
| CN101936989A (en) * | 2010-07-23 | 2011-01-05 | 中国检验检疫科学研究院 | Protein suspension chip for synchronously detecting various antibodies in serum sample and preparation method and using method thereof |
| CN103694358A (en) * | 2013-12-26 | 2014-04-02 | 潍坊医学院 | Locus specificity biotin labeled recombinant IgG (Intravenous Gamma Globulin) affine peptide and application thereof in IgG antibody three-dimensional oriented fixation |
| CN103694358B (en) * | 2013-12-26 | 2015-12-02 | 潍坊医学院 | Locus specificity biotin labeling restructuring IgG affinity peptide and the application fixed at three-dimensional orientation in IgG antibody thereof |
| CN107085109A (en) * | 2017-05-15 | 2017-08-22 | 山东大学深圳研究院 | Application of the high positive charge green fluorescent protein in hepatocarcinoma early diagnosis kit is prepared |
| CN117092344A (en) * | 2023-07-19 | 2023-11-21 | 武汉睿奇生物工程有限公司 | Kit for detecting staphylococcus aureus protein A and application thereof |
| CN117092344B (en) * | 2023-07-19 | 2024-04-19 | 武汉睿奇生物工程有限公司 | Kit for detecting staphylococcus aureus protein A and application thereof |
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Application publication date: 20080326 |