CN101963619A - Method for quantifying and detecting recombinant protein containing green fluorescent protein fusion tag - Google Patents
Method for quantifying and detecting recombinant protein containing green fluorescent protein fusion tag Download PDFInfo
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- CN101963619A CN101963619A CN2010102785130A CN201010278513A CN101963619A CN 101963619 A CN101963619 A CN 101963619A CN 2010102785130 A CN2010102785130 A CN 2010102785130A CN 201010278513 A CN201010278513 A CN 201010278513A CN 101963619 A CN101963619 A CN 101963619A
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Abstract
The invention relates to a method for quantifying and detecting green fluorescent protein or fusion protein containing the green fluorescent protein, which is based on the preparation of green fluorescent protein resisting polyclonal antibody and monoclonal antibody, and can be used for quantifying and detecting the green fluorescent protein by screening paired antibodies and establishing double-antibody sandwich enzyme-linked immunosorbent assay. The method is characterized in that monoclonal anti-coating antibody, specimen and standard substance which are to be tested, polyclone detection antibody, enzyme labeled antibody and substrate are sequentially added into a 96-pore plate, and the light absorption value can be measured when the wavelength is 410nm. The invention overcomes the defects of western blot, can be used for quantifying and detecting, and is strong in specificity, high in sensitivity, good in repeatability as well as simple and convenient to operate, thus being very suitable for quickly quantifying and detecting the green fluorescent protein and the corresponding fusion protein.
Description
Technical field
The present invention relates to a kind ofly utilize the double-antibody sandwich enzyme linked immunosorbent assay to come the detection by quantitative green fluorescent protein or contain the method for the fusion of this albumen.
Background technology
Enter second of 21st century 10 years, human life science has been opened " functional genome's plan " comprehensively, what stand in the breach is exactly the research of proteomics.Protein structure and function diversity have determined that the proteomics research difficulty is big, and the announcement of the 26S Proteasome Structure and Function of new coded by said gene protein is vital to the biologist.No matter be the conventional protein molecular research or the research of protein science, for structure and the function that fully understands albumen, the work requirements of protokaryon or eukaryotic expression recombinant protein molecule increases day by day.Wherein, label protein is a kind of important instrument.Use the advantage that suitable fusion label mainly contains following several respects: one, can increase the stability of destination gene expression product (destination protein), improve expression; Two, improve the solubility of destination protein in the prokaryotic expression system; Three, the label protein of part with the molecular chaperones effect ability that can help destination protein to keep natural structure picture and combine with part; Four, be that detection and/or purifying destination protein are provided convenience.Fusion label commonly used comprises: poly arginine, poly histidine, calmodulin are in conjunction with polypeptide, c-myc, glutathione s-transferase, FLAG polypeptide, staphylococcal protein A, green fluorescent protein, thioredoxin or the like.Particularly green fluorescent protein is with fluorescent characteristic that itself was had and become purposes albumen label very widely.After containing the expressing fusion protein of green fluorescent protein label, can be in qualitative detection under the fluorescent microscope, but still need quantitative detection technique under a lot of situation.At present the detection method to the recombinant protein that has the green fluorescent protein label mainly is semiquantitative Western blot, and this method complicated operation, experimental period are longer, and required reagent is many, the cost height, and can't carry out accurate quantification.
Summary of the invention
In order to overcome the deficiency of Western blot, the invention provides a kind of quantitative detecting method, not only can be quantitative, and high specificity, highly sensitive, good reproducibility, simple and easy to do, be very suitable for the fusion that fast quantification detects green fluorescent protein or contains this albumen.
For reaching above purpose, the present invention takes following technical scheme to be achieved:
A kind of recombinant protein quantitative detecting method that contains the green fluorescent protein fusion tag is characterized in that, comprises the steps:
(1) uses 5mg/L coated antibody bag by 96 orifice plates, place down for 4 ℃ and spend the night;
(2) seal 96 orifice plates with the damping fluid room temperature that contains bovine serum albumin(BSA), discard confining liquid after the washing;
(3) the green fluorescent protein standard items of adding doubling dilution in a part of hole, residue adds sample to be measured in the hole, hatches and washs the back and add detection antibody, and hatch and wash the back again and add enzyme labelled antibody,
(4) hatch and wash the back for the third time with 2,2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) is measured absorbance value as the substrate colour developing in wavelength 410nm;
(5) absorbance value with standard items is an ordinate, and respective concentration is a horizontal ordinate, makes typical curve, according to the absorbance value of sample to be measured, through the curve The Fitting Calculation, draws the concentration of green fluorescent protein in the sample to be measured.
Another kind contains the recombinant protein quantitative detecting method of green fluorescent protein fusion tag,, it is characterized in that, comprise the steps:
(1) uses 5mg/L coated antibody bag by 96 orifice plates, place down for 4 ℃ and spend the night;
(2) seal 96 orifice plates with the damping fluid room temperature that contains bovine serum albumin(BSA), discard confining liquid after the washing;
(3) the green fluorescent protein standard items of adding doubling dilution in a part of hole, residue adds fusion GFP-CD226ICD sample to be measured in the hole, hatches and washs the back and add detection antibody, and hatch and wash the back again and add enzyme labelled antibody,
(4) hatch and wash the back for the third time with 2,2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) is measured absorbance value as the substrate colour developing in wavelength 410nm;
(5) absorbance value with standard items is an ordinate, and respective concentration is a horizontal ordinate, makes typical curve, according to the absorbance value of fusion GFP-CD226ICD to be measured, through the curve The Fitting Calculation, draws the concentration of fusion GFP-CD226ICD to be measured.
In the such scheme, described coated antibody is a monoclonal antibody, its preparation method is: recombinant expressed green fluorescent protein as immunogene, is adopted conventional hybridization knurl method and obtains many strains monoclonal antibody of anti-green fluorescent protein through the anion-exchange chromatography purifying; Described detection antibody is polyclonal antibody, its preparation method is: with recombinant expressed green fluorescent protein as immunogene, adopt routine immunization rabbit method to obtain the polyclonal antiserum of anti-green fluorescent protein, obtain polyclonal antibody through the anion-exchange chromatography purifying, many strains monoclonal antibody need be screened with polyclonal antibody and be matched.
Matching method is: as coated antibody, polyclonal antibody is as detecting antibody with the monoclonal antibody that obtains, and commercial HRP mark goat anti-rabbit antibody adopts the Array Method pairing to select the pairing antibody that susceptibility is the highest and specificity is best as enzyme labelled antibody; Wherein, susceptibility is the highest to be meant, the absorbance value of the green fluorescent protein of detection same concentrations is the highest; Specificity is meant that preferably the absorbance value that detects irrelevant albumen is minimum, also is that non-special background is minimum.
The invention has the beneficial effects as follows that it can detect green fluorescent protein by fast quantification, because this is the method for setting up on the monoclonal antibody basis, and monoclonal antibody is the antibody of the single epi-position of identification, so high specificity of the present invention; Again because screen, so of the present invention highly sensitive through the pairing of antibody; The present invention is based upon on the basis of enzyme linked immunosorbent assay, the whole process of Western blotting needs 2 days approximately, and the present invention only needs earlier with about 5 minutes coated antibodies, be put into do after 4 ℃ of refrigerator overnight the back half, back half about 4 hours, and error is minimum between the absorbance value between each multiple hole, and it is little promptly to criticize a variation within batch coefficient, so the present invention is simple and easy to do in addition, the characteristics of good reproducibility.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment.
The quantitative detecting method of green fluorescent protein utilizes on the basis of anti-green fluorescent protein monoclonal antibody of preparation and polyclonal antibody, through the screening of pairing antibody, sets up the double-antibody sandwich enzyme linked immunosorbent assay, comes the detection by quantitative green fluorescent protein.
Anti-green fluorescent protein MONOCLONAL ANTIBODIES SPECIFIC FOR is: as immunogene, adopt conventional hybridization knurl method to obtain the monoclonal antibody of the anti-green fluorescent protein of many strains recombinant expressed green fluorescent protein.
Anti-green fluorescent protein Polyclonal Antibody Preparation is: as immunogene, adopt routine immunization rabbit method to obtain the polyclonal antiserum of anti-green fluorescent protein recombinant expressed green fluorescent protein, obtain polyclonal antibody through the anion-exchange chromatography purifying.
The screening technique of pairing double antibody is: with the monoclonal antibody that obtains as coated antibody, polyclonal antibody is as detecting antibody, commercial HRP mark goat anti-rabbit antibody adopts the Array Method pairing to select the pairing antibody that susceptibility is the highest and specificity is best, wherein as enzyme labelled antibody; Susceptibility is the highest to be meant, the absorbance value of the green fluorescent protein of detection same concentrations is the highest.Specificity is meant that preferably the absorbance value that detects irrelevant albumen is minimum, also is that non-special background (owing to the combination that nonspecific interaction between albumen causes, showing as the absorbance value of irrelevant albumen in this experimental system) is minimum.
Embodiment 1
The method of detection by quantitative green fluorescent protein:
Recombinant expressed green fluorescent protein as immunogene, is adopted conventional hybridization knurl technology, through Fusion of Cells, screening and cloning, obtain the monoclonal antibody of the anti-green fluorescent protein of 3 strains altogether, respectively called after FMU-GFP3, FMU-GFP4, FMU-GFP5.
As immunogene, adopt routine immunization rabbit technology to obtain the polyclonal antiserum of anti-green fluorescent protein recombinant expressed green fluorescent protein, obtain polyclonal antibody through anion-exchange chromatography technology purifying.
After the pairing of antibody screening, with FMU-GFP3 and FMU-GFP5 simultaneously as coated antibody, with the Na of pH9.6,0.05mol/L
2CO
3-NaHCO
3Damping fluid dilution coated antibody adds 96 orifice plates to 5mg/L, preserves moisture in 100 μ l/ holes, and 4 ℃ of placements are spent the night.Wash 96 orifice plates 3 times with lavation buffer solution, add the damping fluid that contains 0.3% bovine serum albumin(BSA), room temperature sealing 30 minutes.Discard confining liquid, add the green fluorescent protein standard items of doubling dilution in a part of hole, residue adds sample to be measured in the hole, and hatched 1 hour for 37 ℃ in 100 μ l/ holes.Wash plate 3 times, as detecting antibody, use the damping fluid that contains 0.3% bovine serum albumin(BSA) will detect 500 times of antibody dilutions the polyclonal antibody of anti-green fluorescent protein, join in each hole, hatched 1 hour for 37 ℃ in 100 μ l/ holes.Wash plate 3 times, add commercial HRP mark goat anti-rabbit antibody, hatched 1 hour for 37 ℃ in 100 μ l/ holes.Wash plate 3 times, with 2, the colour developing of 2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) substrate, placed 10 minutes for 37 ℃ in 100 μ l/ holes, measures absorbance value in wavelength 410nm.Absorbance value with standard items is an ordinate, and respective concentration is a horizontal ordinate, makes typical curve, and its sensitivity reaches 31.3ng/mL.According to the absorbance value of sample to be measured, through the curve The Fitting Calculation, the concentration that draws green fluorescent protein in the sample to be measured is 500ng/mL.Standard items, the i.e. immunogene (recombinant expressed green fluorescent protein) that is used for immune mouse of concentration known.
Embodiment 2
Detection by quantitative contains the method for the fusion GFP-CD226ICD of green fluorescent protein:
Recombinant expressed green fluorescent protein as immunogene, is adopted conventional hybridization knurl technology, through Fusion of Cells, screening and cloning, obtain the monoclonal antibody of the anti-green fluorescent protein of 3 strains altogether, respectively called after FMU-GFP3, FMU-GFP4, FMU-GFP5.
As immunogene, adopt routine immunization rabbit technology to obtain the polyclonal antiserum of anti-green fluorescent protein recombinant expressed green fluorescent protein, obtain polyclonal antibody through anion-exchange chromatography technology purifying.
After the pairing of antibody screening, with FMU-GFP3 and FMU-GFP5 simultaneously as coated antibody, with the Na of pH9.6,0.05mol/L
2C0
3-NaHCO
3Damping fluid dilution coated antibody adds 96 orifice plates to 5mg/L, preserves moisture in 100 μ l/ holes, and 4 ℃ of placements are spent the night.Wash 96 orifice plates 3 times with lavation buffer solution, add the damping fluid that contains 0.3% bovine serum albumin(BSA), room temperature sealing 30 minutes.Discard confining liquid, add the green fluorescent protein standard items of doubling dilution in a part of hole, residue adds sample GFP-CD226ICD to be measured in the hole, and hatched 1 hour for 37 ℃ in 100 μ l/ holes.Wash plate 3 times, as detecting antibody, use the damping fluid that contains 0.3% bovine serum albumin(BSA) will detect 500 times of antibody dilutions the polyclonal antibody of anti-green fluorescent protein, join in each hole, hatched 1 hour for 37 ℃ in 100 μ l/ holes.Wash plate 3 times, add commercial HRP mark goat anti-rabbit antibody, hatched 1 hour for 37 ℃ in 100 μ l/ holes.Wash plate 3 times, with 2, the colour developing of 2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) substrate, placed 10 minutes for 37 ℃ in 100 μ l/ holes, measures absorbance value in wavelength 410nm.Absorbance value with standard items is an ordinate, and respective concentration is a horizontal ordinate, makes typical curve, and its sensitivity reaches 31.3ng/mL.According to the absorbance value of sample to be measured, after curve match and molecular weight conversion, to calculate, the concentration that draws measured target protein GFP-CD226ICD in the sample to be measured is 300ng/mL.Standard items, the i.e. immunogene (recombinant expressed green fluorescent protein) that is used for immune mouse of concentration known.
Claims (6)
1. a recombinant protein quantitative detecting method that contains the green fluorescent protein fusion tag is characterized in that, comprises the steps:
(1) uses 5mg/L coated antibody bag by 96 orifice plates, place down for 4 ℃ and spend the night;
(2) seal 96 orifice plates with the damping fluid room temperature that contains bovine serum albumin(BSA), discard confining liquid after the washing;
(3) the green fluorescent protein standard items of adding doubling dilution in a part of hole, residue adds sample to be measured in the hole, hatches and washs the back and add detection antibody, and hatch and wash the back again and add enzyme labelled antibody,
(4) hatch and wash the back for the third time with 2,2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) is measured absorbance value as the substrate colour developing in wavelength 410nm;
(5) absorbance value with standard items is an ordinate, and respective concentration is a horizontal ordinate, makes typical curve, according to the absorbance value of sample to be measured, through the curve The Fitting Calculation, draws the concentration of green fluorescent protein in the sample to be measured.
2. the recombinant protein quantitative detecting method that contains the green fluorescent protein fusion tag as claimed in claim 1, it is characterized in that, described coated antibody is a monoclonal antibody, its preparation method is: recombinant expressed green fluorescent protein as immunogene, is adopted conventional hybridization knurl method and obtains many strains monoclonal antibody of anti-green fluorescent protein through the anion-exchange chromatography purifying; Described detection antibody is polyclonal antibody, its preparation method is: with recombinant expressed green fluorescent protein as immunogene, adopt routine immunization rabbit method to obtain the polyclonal antiserum of anti-green fluorescent protein, obtain polyclonal antibody through the anion-exchange chromatography purifying, many strains monoclonal antibody need be screened with polyclonal antibody and be matched.
3. the recombinant protein quantitative detecting method that contains the green fluorescent protein fusion tag as claimed in claim 2, it is characterized in that, described matching method is: with the monoclonal antibody that obtains as coated antibody, polyclonal antibody is as detecting antibody, commercial HRP mark goat anti-rabbit antibody adopts the Array Method pairing to select the pairing antibody that susceptibility is the highest and specificity is best as enzyme labelled antibody; Wherein, susceptibility is the highest to be meant, the absorbance value of the green fluorescent protein of detection same concentrations is the highest; Specificity is meant that preferably the absorbance value that detects irrelevant albumen is minimum, also is that non-special background is minimum.
4. a recombinant protein quantitative detecting method that contains the green fluorescent protein fusion tag is characterized in that, comprises the steps:
(1) uses 5mg/L coated antibody bag by 96 orifice plates, place down for 4 ℃ and spend the night;
(2) seal 96 orifice plates with the damping fluid room temperature that contains bovine serum albumin(BSA), discard confining liquid after the washing;
(3) the green fluorescent protein standard items of adding doubling dilution in a part of hole, residue adds fusion GFP-CD226ICD sample to be measured in the hole, hatches and washs the back and add detection antibody, hatches and washs the back again and add enzyme labelled antibody;
(4) hatch and wash the back for the third time with 2,2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) is measured absorbance value as the substrate colour developing in wavelength 410nm;
(5) absorbance value with standard items is an ordinate, and respective concentration is a horizontal ordinate, makes typical curve, according to the absorbance value of fusion GFP-CD226ICD to be measured, through the curve The Fitting Calculation, draws the concentration of fusion GFP-CD226ICD to be measured.
5. the recombinant protein quantitative detecting method that contains the green fluorescent protein fusion tag as claimed in claim 4, it is characterized in that, described coated antibody is a monoclonal antibody, its preparation method is: recombinant expressed green fluorescent protein as immunogene, is adopted conventional hybridization knurl method and obtains many strains monoclonal antibody of anti-green fluorescent protein through the anion-exchange chromatography purifying; Described detection antibody is polyclonal antibody, its preparation method is: with recombinant expressed green fluorescent protein as immunogene, adopt routine immunization rabbit method to obtain the polyclonal antiserum of anti-green fluorescent protein, obtain polyclonal antibody through the anion-exchange chromatography purifying, many strains monoclonal antibody need be screened with polyclonal antibody and be matched.
6. the recombinant protein quantitative detecting method of green fluorescent protein fusion tag as claimed in claim 5, it is characterized in that, described matching method is: with the monoclonal antibody that obtains as coated antibody, polyclonal antibody is as detecting antibody, commercial HRP mark goat anti-rabbit antibody adopts the Array Method pairing to select the pairing antibody that susceptibility is the highest and specificity is best as enzyme labelled antibody; Wherein, susceptibility is the highest to be meant, the absorbance value of the green fluorescent protein of detection same concentrations is the highest; Specificity is meant that preferably the absorbance value that detects irrelevant albumen is minimum, also is that non-special background is minimum.
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Cited By (7)
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| CN103399156A (en) * | 2013-07-30 | 2013-11-20 | 中国农业科学院北京畜牧兽医研究所 | Green fluorescent protein radio-immunity kit as well as preparation method and detection method thereof |
| CN105273082A (en) * | 2015-09-22 | 2016-01-27 | 北京林业大学 | Method for producing GFP polyclonal antibody |
| CN107290523A (en) * | 2017-05-23 | 2017-10-24 | 西北农林科技大学 | The method that antibody capture albumen and reporter gene fusion recombinant protein detect antibody |
| CN111521813A (en) * | 2020-03-20 | 2020-08-11 | 天德瑞(北京)生物科技有限公司 | Preparation method of green fluorescent protein fusion protein immunoaffinity column, immunoaffinity column and application thereof |
| WO2023123154A1 (en) * | 2021-12-28 | 2023-07-06 | 深圳先进技术研究院 | Method for rapidly constructing sandwich elisa |
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| CN102207504A (en) * | 2011-03-23 | 2011-10-05 | 北京华创远航科技有限公司 | Enzyme-linked immunosorbent assay kit, and preparation method thereof |
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| CN105273082A (en) * | 2015-09-22 | 2016-01-27 | 北京林业大学 | Method for producing GFP polyclonal antibody |
| CN107290523A (en) * | 2017-05-23 | 2017-10-24 | 西北农林科技大学 | The method that antibody capture albumen and reporter gene fusion recombinant protein detect antibody |
| CN111521813A (en) * | 2020-03-20 | 2020-08-11 | 天德瑞(北京)生物科技有限公司 | Preparation method of green fluorescent protein fusion protein immunoaffinity column, immunoaffinity column and application thereof |
| WO2023123154A1 (en) * | 2021-12-28 | 2023-07-06 | 深圳先进技术研究院 | Method for rapidly constructing sandwich elisa |
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Application publication date: 20110202 |