CN101963619A - Method for quantifying and detecting recombinant protein containing green fluorescent protein fusion tag - Google Patents
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- 239000005090 green fluorescent protein Substances 0.000 title claims abstract description 70
- 108010043121 Green Fluorescent Proteins Proteins 0.000 title claims abstract description 56
- 102000004144 Green Fluorescent Proteins Human genes 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims description 26
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims description 12
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims description 12
- 230000012743 protein tagging Effects 0.000 title claims description 10
- 238000001514 detection method Methods 0.000 claims abstract description 32
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- 102000004190 Enzymes Human genes 0.000 claims abstract description 9
- 230000035945 sensitivity Effects 0.000 claims abstract description 7
- 239000000758 substrate Substances 0.000 claims abstract description 7
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 6
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 6
- 230000031700 light absorption Effects 0.000 claims abstract 10
- 239000011248 coating agent Substances 0.000 claims abstract 7
- 238000000576 coating method Methods 0.000 claims abstract 7
- 238000002835 absorbance Methods 0.000 claims description 19
- 238000005406 washing Methods 0.000 claims description 10
- 238000005571 anion exchange chromatography Methods 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- 229940098773 bovine serum albumin Drugs 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 241000283707 Capra Species 0.000 claims description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 6
- 230000003053 immunization Effects 0.000 claims description 6
- 238000002649 immunization Methods 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims 6
- 230000002163 immunogen Effects 0.000 claims 4
- 230000000903 blocking effect Effects 0.000 claims 2
- 210000004408 hybridoma Anatomy 0.000 claims 2
- 238000000746 purification Methods 0.000 claims 2
- 238000003259 recombinant expression Methods 0.000 claims 2
- 239000000243 solution Substances 0.000 claims 2
- 239000000126 substance Substances 0.000 claims 2
- 239000013065 commercial product Substances 0.000 claims 1
- 238000012216 screening Methods 0.000 abstract description 7
- 238000002965 ELISA Methods 0.000 abstract description 4
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- 230000004927 fusion Effects 0.000 description 10
- 238000013016 damping Methods 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 7
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- 238000009396 hybridization Methods 0.000 description 4
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- 230000014509 gene expression Effects 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 2
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- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 102000005431 Molecular Chaperones Human genes 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种绿色荧光蛋白或含该蛋白的融合蛋白的定量检测方法，它利用在制备抗绿色荧光蛋白多克隆抗体和单克隆抗体的基础上，经过配对抗体的筛选，建立的双抗体夹心酶联免疫吸附试验，来定量检测绿色荧光蛋白。其特征是在96孔板中，顺序加入单克隆抗包被抗体、待测标本和标准品、多克隆检测抗体、酶标抗体及底物，于波长410nm测定光吸收值。本发明克服了免疫印迹法的不足，不仅可以定量检测，而且特异性强、灵敏度高、重复性好、简便易行，非常适合于快速定量检测绿色荧光蛋白及相应的融合白。The invention relates to a quantitative detection method for green fluorescent protein or a fusion protein containing the protein, which utilizes a double-antibody sandwich established on the basis of preparing polyclonal antibodies and monoclonal antibodies against green fluorescent protein and screening paired antibodies Enzyme-linked immunosorbent assay for quantitative detection of green fluorescent protein. It is characterized in that monoclonal anti-coating antibody, test specimen and standard product, polyclonal detection antibody, enzyme-labeled antibody and substrate are sequentially added to the 96-well plate, and the light absorption value is measured at a wavelength of 410nm. The invention overcomes the disadvantages of immunoblotting, not only can quantitatively detect, but also has strong specificity, high sensitivity, good repeatability, is simple and easy to implement, and is very suitable for rapid quantitative detection of green fluorescent protein and corresponding fusion proteins.
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.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN102426240A (en) * | 2011-09-19 | 2012-04-25 | 北京华创远航科技有限公司 | Enzyme-linked detection kit and preparation method thereof |
| 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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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102426240A (en) * | 2011-09-19 | 2012-04-25 | 北京华创远航科技有限公司 | Enzyme-linked detection kit and preparation method thereof |
| CN102426240B (en) * | 2011-09-19 | 2014-10-15 | 北京健平金星生物科技有限公司 | Enzyme-linked detection kit and preparation method thereof |
| 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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