CN102364342A - Method for quickly detecting expression of recombinant protein - Google Patents
Method for quickly detecting expression of recombinant protein Download PDFInfo
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- CN102364342A CN102364342A CN2011102234537A CN201110223453A CN102364342A CN 102364342 A CN102364342 A CN 102364342A CN 2011102234537 A CN2011102234537 A CN 2011102234537A CN 201110223453 A CN201110223453 A CN 201110223453A CN 102364342 A CN102364342 A CN 102364342A
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Abstract
The invention relates to a method for quickly detecting the expression of a recombinant protein, which is characterized in that: an immune colloidal gold technique is used for conveniently and quickly detecting whether a recombinant protein exists in a sample. The method can be used for the expression experiment of the recombinant protein in relevant scientific research of biological medicine and can also be used for frequently detecting and quickly semi-quantitatively estimating an expression level of the recombinant protein during the fermentation production process of recombinant protein medicine in the biological pharmaceutical enterprises. In the application, the method can partially substitute the conventional sodium dodecyl sulfate (SDS) gel electrophoresis and immunological imprinting detection, so the detection procedure is remarkably simplified, the detection time is shortened, and the detection cost is reduced.
Description
One technical field
The present invention relates to a kind of immune colloid gold coupling monoclonal antibody method and application; A kind of method for quick that gene recombinant protein is expressed that is applied to is provided; This method utilization is to the specific antibody of specific antigen epi-position (can be label or non-label composition) in the recombinant expression protein; Can be easy/differentiate whether there is expressed target proteins in the biological sample apace, whole testing process is no more than 30 minutes.
Two background technologies
Gene recombinant expressed become a kind of conventional means in the life science technology, is applied to the every field of life science and production, like the functional study of protein, biological medicine research, production of medicine or the like.The detection of exogenous gene expression for ease in gene recombinant expressed; Most of albumen are to express with the form of fusion; Just add some and have the peptide sequence of epitope character at the aminoterminal of desiring expressing protein or c-terminus; Promptly express label, detect expressing sample, can confirm whether foreign gene expresses through using the label specific antibody.Expression label commonly used has: GST label, His label, c-myc label, V5 label, HA label, Trx label, GFP label and FLAG label or the like.Some can also be used to that expressing protein is carried out purifying such as GST label and His label and just can be respectively with the gel of glutathione and nickel coupling expressing protein be carried out affinity purification in these labels commonly used.
In the research and production of dna recombinant expression, whether the testing goal gene expresses in host's (bacterium, fungi, cell, tissue, organ) is vital.Detection method commonly used has: for the higher prokaryotic expression system of expression; Can use conventional SDS-PAGE to detect; With do not induce or do not have the bacterium that inserts fragment and compare, have significantly new band to occur, promptly tentatively thinking has the expression of genes of interest.For expression lower prokaryotic expression carrier and eukaryotic expression system; Be difficult to see the obvious expression band after the SDS-PAGE dyeing; Therefore need detect through the higher Western blotting of sensitivity (Western Blot) method sample, whether express to confirm genes of interest.
Immune colloidal gold technique has been widely used in biomedical sector since the eighties of last century invention seventies.Compare the characteristics that immune colloid gold reagent has is easy, fast, do not rely on any instrument with traditional immune reagent for clinical diagnosis.The principle of immune colloid gold reagent is through nanogold particle being marked on the specific antigen and antibody; After the antibody that is labeled or antigen and immobilised corresponding antigen or antibody react; Cause the gathering of the antibody that is labeled or antigen, thereby produce macroscopic red stripes at regional area.Compare with traditional SDS-PAGE and Western Blot, that immune colloidal gold technique has is easy, fast, do not rely on characteristics such as any instrument: easy, the sample preparation scheme is easy, does not need processes such as electrophoresis; Fast, Western Blot needs 2 day time could detect completion, and SDS-PAGE also needs 4---and 5 hours, and the immune colloidal gold technique overall process that the present invention adopts is no more than 30 minutes; Do not rely on any instrument, SDS-PAGE needs electrophoresis apparatus and corresponding buffer system, and the instrument reagent that Western Blot then needs is more, and immune colloidal gold technique is then without any need for instrument, and the simple buffering system that only needs gets final product.Therefore, the present invention is fit to the fast detecting of recombination fusion protein very much, for the plenty of time has been saved in scientific research research, protein expression production etc.
Three summary of the invention
The objective of the invention is to provide a kind of specific antibody and colloidal gold technique utilized, the easy method of gene recombinant protein expression and the related reagent that is used to realize this purpose that method produced thus of detecting apace.The recombinant expressed of gene can be that native protein is expressed, and also can be the fusion that has label.When the specific antibody that uses to recombinant expression protein self composition, the used method of the present invention with according to the reagent of these methods preparations can and only to be used for the detection of this kind expression of recombinant proteins.When using the specific antibody that is directed against label composition in the recombination, amalgamation and expression albumen; The used method of the present invention and the reagent according to these methods preparations can be used as a kind of testing product of universality, are used to detect any Expression of Fusion Protein that has this label.The recombination fusion protein label that is suitable in the methods of the invention includes but not limited to 1) the amalgamation and expression albumen that has the His label of in prokaryotic system and eukaryotic system, expressing; The amalgamation and expression albumen that has the GST label of 2) in prokaryotic system and eukaryotic system, expressing; The amalgamation and expression albumen that has the TRX label of 3) in prokaryotic system and eukaryotic system, expressing; The amalgamation and expression albumen that has the V5 label of 4) in prokaryotic system and eukaryotic system, expressing; The amalgamation and expression albumen that has the c-myc label of 5) in prokaryotic system and eukaryotic system, expressing; The amalgamation and expression albumen that has the FLAG label of the amalgamation and expression albumen 8 that has the GFP label of the amalgamation and expression albumen that has the HA label of 6) in prokaryotic system and eukaryotic system, expressing, 7) in prokaryotic system and eukaryotic system, expressing) in prokaryotic system and eukaryotic system, expressing.The invention provides the concrete grammar of realizing above-mentioned target, comprising: 1) selection and the purifying of specificity recombinant protein antibody (to label or the non-label composition in the recombinant protein); 2) preparation method of nanogold particle; 3) with the method for nanogold particle labelled antibody; 4) preparation of immune colloid gold quick detection test paper bar and assemble method; 5) use immune colloid gold quick detection test paper bar to detect the method for recombinant protein; 6) detection method of invention correlation parameter
Involved in the present invention be intended to simplify reorganization tag fusion protein detection of expression step; Shorten the survey time; Improve the method for detection efficiency, mainly may further comprise the steps: 1) selection and the purifying of specificity recombinant protein antibody (to label or the non-label composition in the recombinant protein), the difference of the recombinant protein that detects according to desire is selected the antibody of the recombinant protein of the required detection of specific recognition for use; This antibody can be polyclonal antibody, also can be monoclonal antibody.Selected antibody need carry out purifying, and the antibody purification method of the routine that the method for purifying can be selected to use at present includes but not limited to: sad-ammonium sulfate precipitation method, Protein A or Protein G affinity purification, and the antigen affinity purification etc.2) preparation of the antibody of nano gold mark prepares the nm of gold colloidal solution of certain diameter through reducing process.The diameter of used colloid gold particle is 20nm among the present invention, on the basis of existing method, changes a little to prepare the colloid gold particle of diameter range between 10-200nm.Antibody after using this colloidal gold solution to purifying then carries out mark, and the antibody behind the mark is through being used for the preparation of immunity colloidal gold test paper strip behind the purifying.3) preparation of immune colloid gold quick detection test paper bar and assemble method.4) use immune colloid gold quick detection test paper bar to detect the method for recombinant protein.
Recombinant Protein Expression is the experimental technique of current biology and the widespread use of medical scientific research association area.The present invention utilizes immune colloidal gold technique that the recombinant protein that includes but not limited to express in protokaryon and the eucaryote is carried out easy detection apace, and part replaces conventional sds gel electrophoresis and the immune marking detects, and has improved work efficiency significantly.In field of biological pharmacy, in the fermentation production process of recombinant protein medicine, need frequent detect the Recombinant Protein Expression level with regulation technology parameter correspondingly, confirm fermentation termination.The inventive method can the sxemiquantitative mode be carried out rapid evaluation to the expression of recombinant protein, has simplified trace routine, has shortened detection time and has reduced the detection cost.
Four. description of drawings
Accompanying drawing 1 colloidal gold solution quality testing
Accompanying drawing 2 collaurum optimal pH conditions are groped
Accompanying drawing 3 collaurum optimal pH conditions are confirmed
Accompanying drawing 4 collaurum optimum antibody label concentration are confirmed
Accompanying drawing 5 collaurum cross reactions detect.Has only T heading line off when containing the His tag fusion protein in the sample.In sample, do not have recombinant protein, or recombinant protein when not containing the His label T line occur.The C line all occurs in each pattern detection, and the prompting detectable is in proper working order, and testing result is effective.
Accompanying drawing 6 collaurum sensitivity detect.When the T heading line off during of His tag fusion protein content in the sample more than or equal to 1 μ g/ml.The T line occurs when His tag fusion protein content is less than or equal to 100ng/ml in the sample.The C line all occurs in each pattern detection, and the prompting detectable is in proper working order, and testing result is effective.
Five. embodiment
Below be example with preparation and the test experience of reorganization His tag fusion protein quick detection test paper, the implementation process and the actual applicability of description the inventive method.These embodiment also do not limit the present invention in any way the scope of the claim that awaits the reply.The purifying of embodiment 1. anti-His tag monoclonal antibodies:
The required reagent of monoclonal antibody purifying: a) the Sepharose gel of Protein G coupling (GE Company products), b) phosphate buffer (PBS): 20mM sodium phosphate, 0.15M NaCl, pH 7.2, c) eluent: Glycin-HCl (pH 2.7)
The monoclonal antibody of mouse anti His label is accomplished through conventional cell-fusion techniques preparation for the reagent bio tech ltd by Beijing health, and the monoclonal antibody of anti-His label obtains from mouse ascites through the method for Protein G affinity purification.Get 10ml ascites; After 1 times of PBS (pH7.2) dilution; Add the Sepharose gel of 2ml Protein G coupling is housed, control flow velocity 0.2ml/min is after OD 280 monitor values that wash affinity column to effluent with level pad PBS after last appearance is accomplished are got back to baseline values; With the Glycin-HCL wash-out antibody of pH2.7, the solution behind the wash-out neutralizes with 1M Tris immediately.Antibody after the purification detects its purity through SDS-PAGE, and measures its antibody activity with the ELISA method.
The preparation of embodiment 2. nm of gold colloidal solution:
The employing trisodium citrate reduction method also is summarized as follows: get the triangular flask of silication, add 100mL deionized water and 1mL1% gold chloride, ebuillition of heated; 1% trisodium citrate that adds rapidly 4ml can be observed the very fast grizzle of flaxen aqueous solution of chloraurate this moment, and is continuous and change into black, stablizes gradually subsequently to become red.The about 3min of overall process continues to boil 15min, and volume is supplied to 100mL with deionized water in the cooling back.Preparation effect with visible light scanning detection collaurum has single absorption peak (accompanying drawing 1) at the 522nm place.
The method of embodiment 3.His tag antibody marking nano gold grain
3.1 the optimum pH that antibody combines with collaurum is confirmed
Get the 1.5mL centrifuge tube, add the 1mL colloidal gold solution respectively, the pH value is transferred to 6.0,6.5,7.0,7.5,8.0,8.5,9.0 and 9.5 respectively with 0.1mol/L K2CO3.Monoclonal antibody solution with 4 ℃ of centrifugal 20min of 3000r/min, is removed insoluble sludge.The monoclonal antibody of getting 50 μ L 1mg/mL adds in the above-mentioned collaurum pipe, and room temperature is placed 10min behind the concussion 20min; Every then pipe adds 100 μ L 10%NaCl solution respectively, and concussion mixes the back room temperature and places 10min, carries out visible light scanning and detects, and writes down the highest pH value X of coupling rate (the pH value when under the same conditions, absorption peak is the highest).Again the pH value is transferred to X-0.6, X-0.4, X-0.2, X, X+0.2, X+0.4 and X+0.6, repeat above-mentioned test, the pH value that the coupling rate is the highest is optimum pH.The optimization experiment result of pH condition shows that suitable pH scope is between 6.4 and 10.0, and pH scope preferably is between 7.0 and 8.0, and optimum pH condition is pH7.4.The visible spectrum scanning detecting result is seen accompanying drawing 2 and 3.
3.2 confirming of the optium concentration that antibody combines with collaurum
The collaurum of getting 0.5mL pH7.4 respectively joins in the 1.5mL centrifuge tube, adds His monoclonal antibody solution, makes its final concentration be respectively 0,15,20,25,30,35,40,45 μ g/mL, and concussion mixes 20min, and room temperature is placed 10min.Every then pipe adds 100 μ L100%NaCl; Concussion mixes the back room temperature and places back 10min; Carry out visible light scanning and detect, the result shows that suitable antibody final concentration scope is between the 15-45 μ g/mL; Antibody final concentration scope preferably is between the 35-45 μ g/mL, and optimum AC is 35 μ g/mL (accompanying drawings 4).
3.3 the preparation of colloidal gold probe and purifying
Get the 1.5mL centrifuge tube, add the 1mL collaurum; With 0.1mol/L K2CO3 adjust pH optimal pH; Dropwise add the His monoclonal antibody, the about 5min of whole process adds.Shake up 15～20min, room temperature is placed 10～15min; Add 10%BSA to final concentration be 1.5% (the mark volume can increase, and needed corresponding reagent also increases)
The colloidal gold antibody solution of mark is used differential centrifugation and is carried out purifying: the colloidal gold solution of mark with the centrifugal 45s of 10000r/min, is discarded deposition.The centrifugal 30min of 12000r/min inhales and abandons supernatant; The borate buffer that adds 1/10 volume, abundant dissolution precipitation, repeated centrifugation 2～3 times forwards in the 1.5mL centrifuge tube.The colloidal gold probe that purifying is good is with the borate buffer dissolving of equivalent.
The preparation and the assembling of embodiment 4. immunity colloidal gold test paper strips
4.1 the preparation of spun glass membrane probe
Amount by 0.5mL/ bar (0.5 * 5cm/ bar) is uniformly coated on colloidal gold probe on the spun glass, places superclean bench aeration-drying to spend the night, and 37 ℃ of placements are subsequent use.
4.2NC the pre-service of film
With the NC film, with 37 ℃ of sealings of 0.01mol/L PBS+1%BSA+0.2%Tween-20+0.05mol/LNaCl solution, 30～60min, deionization washing 2 times, 4 ℃ of dried overnight are subsequent use.
4.3T the optimization of line and C lines spare
Several groups of different antigens concentration are set the T line (detection line) and the C line (nature controlling line) of NC film is optimized, optimize one group of concentration that encapsulates, be respectively 1.0-20.0mg/mL and 1.0mg/mL as T line and C line.The T line encapsulate the detection sensitivity that concentration will determine the test strips product.Prepare a series of T lines and encapsulate the concentration difference, thereby the different test strips of detection sensitivity can form the test strip suit of tool half-quantitative detection function.T line and C line width are 0.5mm.
4.4T stroke film method of line and C line
According to the results of optimization of T line and C line antigen concentration, respectively that purifying is good antigen and sheep anti-mouse igg antibody are diluted to optium concentration with damping fluid; It is subsequent use after 4 ℃ of placements of NC film are spent the night.
4.5 the assembling of test paper
Put on one's gloves, the NC film that encapsulates is adhered on the PVC base plate, the lower edge of attention film aligns careful floating face with the mark line on the mould.The spun glass of the good golden labeling antibody of spraying is adhered on the PVC base plate near the scale lower limb, and carefully floating.Sample pad is adhered on the PVC base plate near the mould lower limb, and carefully floating.Thieving paper is adhered on the PVC base plate near the mould coboundary, and carefully floating.Be cut into the wide test paper of 3.5mm with cutting cutter, the test paper that cuts put into the commodity plastic casing of appropriate size and placed the packaging bag that drying agent is housed to preserve in the assembly section.
The method of application and the result of embodiment 5. immunity colloidal gold test paper strip fast detecting reorganization His tag fusion protein judge
The test strips well is upwards placed flat surface, in well, splash into about 100 microlitre samples to be tested.Observe testing result after leaving standstill 5～10min under the room temperature.Two red lines (C line and T line) appear in negative reaction, do not contain reorganization His tag fusion protein in the prompting sample.A red line (C line) only appears in positive reaction, contains reorganization His tag fusion protein in the prompting sample and detects effective.Nature controlling line (C line) must occur, otherwise this test paper invalid (accompanying drawing 5).
Embodiment 6. invention correlation parameter experiments detect
6.1 specificity experiment
Collect the Escherichia coli bacteria liquid and the Escherichia coli bacteria liquid that contains non-His label recombinant protein that contain reorganization His tag fusion protein respectively; Compare with the Escherichia coli bacteria liquid that does not contain any recombinant protein in addition; With the operation of commodity bacterial lysate (Beijing health is a century bio tech ltd product) by specification, the method with embodiment 5. after the bacterium cracking is detected.Experimental result shows to have only T heading line off when containing the His tag fusion protein in the sample.In sample, do not have recombinant protein, or recombinant protein when not containing the His label T line occur.The C line all occurs in each pattern detection, and the prompting detectable is in proper working order, and testing result is (accompanying drawing 5) effectively.The test paper of testing result explanation preparation can effectively detect the recombination fusion protein that contains the His label, and not with non-His label bacterium liquid generation cross reaction.
6.2 sensitivity experiment
Described in embodiment 4.3, the T line encapsulate the detection sensitivity that concentration will determine the test strips product.Prepare a series of T lines and encapsulate the concentration difference, thereby the different test strips of detection sensitivity can form the test strip suit of tool half-quantitative detection function.This experiment is the sensitivity of the test strips demonstration product of 1.0mg/mL with T line package amount.
The His tag fusion protein of will recombinating detects with product of the present invention after being diluted to 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 100ng/ml, 10ng/ml, 1ng/ml.The result shows, when the T heading line off during more than or equal to 1 μ g/ml of His tag fusion protein content in the sample.The T line occurs when His tag fusion protein content is less than or equal to 100ng/ml in the sample.The C line all occurs in each pattern detection, and the prompting detectable is in proper working order, and testing result is effective.Detection sensitivity is about 1 μ g/ml.(accompanying drawing 6).
6.3 accuracy rate detects
42 Escherichia coli bacteria liquids (SDS electrophoresis detection result shows 8 feminine genders, 34 positives) that have the His label to picked at random are invented finished product detection with this: 8 negative bacterium liquid testing results are all negative; 32 positive bacteria liquid testing results are positive, and the testing result of 2 positive bacteria liquid is negative, and accuracy rate is 94%.
Claims (6)
1. one kind is utilized immune colloidal gold technique to identify in the sample whether have the reorganization tag fusion protein, and can carry out the method for half-quantitative detection to the reorganization tag fusion protein.Comprise the concrete grammar of realizing above-mentioned target: label protein Purification of Monoclonal Antibodies, the preparation of colloid gold reagent and test strips, concrete detection method and the decision method of testing result.
2. according to the method for claim 1, the fusion label that the method capable of using detects includes but not limited to label commonly used, as, GST label, His label, c-myc label, V5 label, HA label, Trx label, GFP label, and the FLAG label etc.
3. according to the method for claim 1; When using the specific antibody that is directed against recombinant expression protein self composition; The method capable of using detects the expression of recombinant proteins situation that does not contain label, includes but not limited to the recombinant protein of in eucaryon recombinant expression system and prokaryotic expression system, expressing.
4. according to claim 1,2,3 method, the application that in the relevant research and development of biological medicine, expression of recombinant proteins is detected.
5. according to claim 1,2,3 method, in the fermentation production process of recombinant protein medicine to detection and sxemiquantitative assessment and other application of recombinant protein expression.
According to claim 1,2,3 method in the commerce that includes but not limited to industries such as pharmacy, animal doctor, veterinary drug, food, feed, biochemical and molecular biology reagent and the application of production field.
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| CN106834319A (en) * | 2017-02-28 | 2017-06-13 | 中南大学湘雅医院 | A kind of preparation method of the coded sequence of recombinant protein, recombinant protein and its monoclonal antibody |
| CN106890622A (en) * | 2017-02-23 | 2017-06-27 | 南昌大佳科技有限公司 | Affine sorbing material based on anti-c Myc label nano antibodies |
| CN108893487A (en) * | 2018-07-19 | 2018-11-27 | 中国农业科学院北京畜牧兽医研究所 | A kind of construction method of plant expression plasmid carrier containing C-Myc protein fusion label and its carrier |
| CN109336978A (en) * | 2018-10-24 | 2019-02-15 | 南京融捷康生物科技有限公司 | Application of the specificity for the single domain antibody of V5 label protein |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106890622A (en) * | 2017-02-23 | 2017-06-27 | 南昌大佳科技有限公司 | Affine sorbing material based on anti-c Myc label nano antibodies |
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| CN109336978A (en) * | 2018-10-24 | 2019-02-15 | 南京融捷康生物科技有限公司 | Application of the specificity for the single domain antibody of V5 label protein |
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Application publication date: 20120229 |