CN105044047A - Kit for detecting recombinant protein expression and using method thereof - Google Patents
Kit for detecting recombinant protein expression and using method thereof Download PDFInfo
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- CN105044047A CN105044047A CN201510312076.2A CN201510312076A CN105044047A CN 105044047 A CN105044047 A CN 105044047A CN 201510312076 A CN201510312076 A CN 201510312076A CN 105044047 A CN105044047 A CN 105044047A
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Abstract
The invention provides a kit for detecting recombinant protein expression and a using method of the kit. The kit comprises a receptor microsphere coupled with an anti-His-tag antibody, a biotin-labeled polyclonal antibody, and an avidin-labeled donor sphere. The kit is used for detecting recombinant protein expression quantity based on antigen-antibody specific binding, a biotin-avidin amplification system, and a reactive oxygen transfer homogeneous phase luminescence detection system. A receptor sphere-His-tag Ab, breaking bacterial liquid with His-tag target protein, and Bio-anti-target protein Ab are mixed and incubated, if the target protein is successfully expressed, a receptor sphere-His-tag Ab-target protein-Bio-anti-target protein Ab compound can be formed, then the avidin-labeled donor sphere is added, avidin is bound with biotin, energy transfer is excited by utilizing reactive oxygen to produce a luminescence phenomenon, and a signal value is read through an instrument.
Description
Technical field
The invention belongs to gene engineering technology field, for detecting recombinant antigen expression, especially relating to a kind of kit and the using method thereof that detect expression of recombinant proteins situation.
Background technology
DNA recombinant technique is an important branch of modern biology development, is the outstanding field of Development of Molecular Biology, greatly facilitates the progress of life science Study and appliance.Wherein, gene constructed on expression vector by certain destination protein, utilize certain biological bacterium or cell to carry out great expression destination protein, the namely expression of recombinant protein is one of important application of DNA recombinant technique.The appearance of expression of recombinant proteins technology make people can utilize engineered means produce required for amino acid sequence (protein), and then the protein that a large amount of acquisition biosome of being difficult in the past obtain is inside and outside, also be Study on Protein functional structure simultaneously, and the powerful of screening targeted drug, play vital role at basic science and medicine.
Current expression of recombinant proteins system has many, and the difference according to the host of expressing protein can be divided into prokaryotic expression system, eukaryotic expression system.Different expression systems has respective relative merits, usually selects according to the character of destination protein and follow-up application.Prokaryotic expression system utilizes specific colibacillus engineering strain as Host Strains to express a system of foreign recombinant proteins, because its genetic background is clear, cultivate simple to operate, transformation efficiency is high, growth and breeding is fast, with low cost, and the advantages such as destination protein can be produced in rapid large-scale and be widely used in the expression of foreign protein.At present, most expression vector often with His label, mainly plays a role in protein purification after expression.In essence, His label is a kind of compatibility label, and affine with IMAC (immobilization metal chelating affinity chromatography), the sequencing more that the purge process of destination protein is become, has more controllability.
Expression of recombinant proteins amount is subject to the impact of many factors, as abduction delivering time, cultivation temperature, IPTG concentration etc.The number of expression of recombinant proteins amount is very crucial to follow-up work.Therefore, a kind of method setting up Quantitative detection expression of recombinant proteins amount is conducive to increasing work efficiency and accuracy.Method mainly polyacrylamide gel electrophoresis (SDS-PAGE) and the western blot test (western-blot) of current detection recombinant protein.And SDS-PAGE can only estimate the relative prevalence of recombinant protein expression, can not accurate quantitative analysis, and need certain expression to identify by naked eyes.Western blot test transfers on nitrocellulose membrane by protein band after electrophoretic separation, after recycling antigen and corresponding antibody carry out immune response, by corresponding chromogenic reaction, to detect the expression of corresponding destination protein.Above-mentioned recombinant protein Measures compare is consuming time, and complex operation is not suitable for being applied to mass detection.In addition, restructuring luciferase and green fluorescent protein is utilized to detect expression of recombinant proteins amount also more common.Green fluorescent protein (greenfluorescentprotein), is called for short GFP, and this protein the earliest Shi Youxia people from village such as to be repaiied and found in the jellyfish of a kind of formal name used at school Aequoreavictoria in 1962.The protein that its gene produces, under the light of blue wavelength region excites, can green fluorescent be sent, detect recombinant protein with this, the help of cold light a-protein equorin is also needed in the process of this luminescence, and this cold light protein and calcium ion (Ca
2+) can reciprocation be produced.Luciferase (Luciferase) is the general designation that occurring in nature can produce the enzyme of biological fluorescent, in corresponding chemical reaction, the generation of fluorescent is the oxidation coming from fluorescein, also comprises atriphos (ATP) in some cases in reaction system.These two kinds of method costs are higher, unstable, can not accurate quantitative analysis, and easily cover downstream Small molecular destination protein, reduce detection accuracy, even affect the biologic activity of recombinant protein.
Summary of the invention
In view of this, the present invention is intended to propose a kind of kit and the using method thereof that detect expression of recombinant proteins situation, to realize the object of Quantitative detection destination protein.
For achieving the above object, the technical scheme of the invention is achieved in that
Detect a kit for expression of recombinant proteins situation, comprise the donor ball of the acceptor microballoon of coupling anti-His-tag antibody, the biotin labeled polyclonal antibody for destination protein, Avidin mark; Preferably, biotinylated antibody dilution, acceptor microballoon dilution is also comprised.Described biotin labeled polyclonal antibody, for destination protein antibody.
Preferably, also comprise luminescence-producing reaction plate, preferably, described luminescence-producing reaction plate is the luminous check-out consoles in 96 holes.
Present invention also offers and use the kit detecting expression of recombinant proteins situation as above to carry out the method detected, comprise following operation steps,
1) Escherichia coli after abduction delivering are carried out ultrasonication, namely obtain the broken bacterium liquid (sample to be checked) with His label destination protein;
2) by the acceptor ball solution of anti-for coupling His-tag antibody, add the mixing of luminescence-producing reaction plate with the broken bacterium liquid of His label destination protein, the Anti-TNF-α liquid solution of biotin labeled anti-destination protein, do 2 ~ 4 multiple holes, incubation 0.5 ~ 1.5 hour at 37 DEG C, preferably, incubation 1 hour; Carry out negative control simultaneously, the operation of described negative control is that the ultrasonication bacterium liquid of the acceptor ball solution of anti-for coupling His-tag antibody, non-abduction delivering, the Anti-TNF-α liquid solution of biotin labeled anti-destination protein are added the mixing of luminescence-producing reaction plate, do 2 ~ 4 multiple holes, heated culture temperature and the time identical with the incubation of the broken bacterium liquid with His label destination protein;
3) under lucifuge condition, the donor ball solution of Avidin mark is added to each hole, incubation 10 ~ 20min at 37 DEG C; Preferably, 15min;
4) reading, result judges; As the signal value of signal value >=2 negative control of bacterium liquid to be detected, be then determined with the great expression of recombinant protein, and signal value is larger, expression level is higher; Preferably, by light stimulated luminescence detector sensed light signal under 615nm.
The present invention adopts the state-of-the-art homogeneous luminescent immunoassay system based on the transfer of active oxygen excitation energy at present, namely detects expression of recombinant proteins amount based on antigen and antibody specific combination and biotin-avidin amplification system.By acceptor ball-His-tagAb (the acceptor ball of coupling anti-His-tag antibody), hatch with the broken bacterium liquid of His-label destination protein and Bio-anti-destination protein Ab (polyclonal antibody of biotin labeled anti-destination protein) mixing, if destination protein successful expression, then can form acceptor ball-His-tagAb-destination protein-Bio-anti-destination protein Ab compound, add the donor ball of Avidin mark again, then Avidin is combined with biotin, active oxygen excitation energy is utilized to shift, produce luminescence phenomenon, read signal value by instrument.
Preferably, step 1) in, with the OD of the broken bacterium liquid of His label destination protein
600nmbe 0.1 ~ 0.5.
Preferably, described step 2) in, the acceptor ball solution of coupling anti-His-tag antibody, be 1:1:1 with the volume ratio of the broken bacterium liquid of His label destination protein, the Anti-TNF-α liquid solution of biotin labeled anti-destination protein.
Preferably, the acceptor ball solution of described coupling anti-His-tag antibody be the acceptor ball of the anti-His-tag antibody of coupling and acceptor dilution by volume 1:50 carry out allocating, preferably, acceptor dilution is Tris-HCl damping fluid, its pH is 8.0, and concentration is 0.1mol/L.
Preferably, the Anti-TNF-α liquid solution of described biotin labeled anti-destination protein be the polyclonal antibody of biotin labeled anti-destination protein and biotin antibody dilution by volume 1:2000 carry out allocating; Preferably, described biotin antibody dilution is PBS damping fluid, and its pH is 7.4, and concentration is 0.1mol/L.
Preferably, step 3) in, the donor ball solution of the Avidin mark added to each hole is 1:7 with the volume ratio with the broken bacterium liquid of His label destination protein, and preferably, the concentration of the donor ball solution of Avidin mark is 20 mcg/ml.
Invention also provides kit as above and detect the application in expression of recombinant proteins.
Relative to prior art, kit of the present invention, has following advantage:
(1) twice accumulative 75min of temperature bath simple to operate, whole testing process is no more than 90min, and conventional western blot test at least needs 2 working days (48h).Particularly this method belongs to homogeneous luminescent immunoassay system, and whole testing process is without " washing " link, and detection efficiency is higher, and precision is stronger.
(2) susceptibility height adopts the luminescence immunoassay system transmitted based on active oxygen, there is higher susceptibility, component to be checked does not need purifies and separates, just can the expression of destination protein of this bacterial strain of Accurate Determining, can realize the Quantitative detection to recombinant protein expression.
(3) in the stronger native system of versatility, the acceptor microballoon of coupling anti-His-tag antibody, the donor ball that marks with Avidin are general detection reagent, replacing biotin labelled antibodies is applicable to all expression systems carrying His-tag expression plasmid, as long as can have the detection of any destination protein.And biotin labelled antibodies convenient sources, even if need in person to mark also very simple.
Accompanying drawing explanation
The accompanying drawing forming a part of the present invention is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is that recombinant protein quantitatively detects homogeneous luminescent immunoassay principle schematic;
Description of reference numerals:
1-destination protein to be checked, 2-His label, the acceptor ball of the anti-His-tag antibody of 3-coupling, the polyclonal antibody of the biotin labeled anti-destination protein of 4-, the donor ball of 5-Avidin mark.
Embodiment
Below with reference to the accompanying drawings and describe the present invention in detail in conjunction with the embodiments.
Embodiment one
The composition of kit: the 96 luminous check-out consoles in holes, for the acceptor microballoon of the anti-His-tag antibody of coupling and the donor ball of Avidin mark purchased from Bo Yang biotechnology (Shanghai) Co., Ltd.; Biotin labeled polyclonal antibody purchased from American sigma company; Damping fluid is commonly used in biotinylated antibody dilution and the equal laboratory of acceptor microballoon dilution, and described biotin antibody dilution is PBS damping fluid, and its pH is 7.4, and concentration is 0.1mol/L; Acceptor dilution is Tris-HCl damping fluid, and its pH is 8.0, and concentration is 0.1mol/L
To be detected as example at prokaryotic expression system-expression in escherichia coli recombinant protein beta lactoglobulin, concrete grammar is as follows:
(1) preparatory stage: the bacterium liquid of abduction delivering beta lactoglobulin is carried out ultrasonication, OD is adjusted
600nmbetween=0.1 ~ 0.5, the bacterium liquid of non-abduction delivering is done same process simultaneously; The acceptor microballoon acceptor microspheres solution 1:50 of anti-for coupling His-tag antibody is diluted, for subsequent use; Anti-for biotin labeling beta lactoglobulin polyclonal antibody biotinylated antibody solution 1:2000 is diluted, for subsequent use.
(2) broken to 25 μ l acceptor ball-His-tagAb (1:50 dilution), 25 μ l bacterium liquid (OD600nm=0.1 ~ 0.5) and the anti-beta-lactoglobulin antibody of 25 μ lBio-(1:2000 dilution) are added the luminous check-out console mixing in 96 holes incubation, 37 DEG C, 1 hour, the ultrasonication bacterium liquid of non-abduction delivering is done negative control simultaneously, do 3 multiple holes respectively.
(3) under lucifuge condition, the donor ball solution of 175 μ l Avidin marks is added to each hole, 37 DEG C of incubation 15min.The concentration of the donor ball solution of Avidin mark is 20 mcg/ml.
(4) reading, result judgement light stimulated luminescence detector sensed light signal under 615nm.Bacterium liquid signal value to be detected is 695, and negative control signal value is 165, detected value >=2 negative control, so result of determination is positive, namely has the great expression of recombinant protein.
The foregoing is only the preferred embodiment of the invention; not in order to limit the invention; within all spirit in the invention and principle, any amendment done, equivalent replacement, improvement etc., within the protection domain that all should be included in the invention.
Claims (9)
1. detect a kit for expression of recombinant proteins situation, it is characterized in that: the donor ball comprising the acceptor microballoon of coupling anti-His-tag antibody, biotin labeled polyclonal antibody, Avidin mark; Preferably, biotinylated antibody dilution, acceptor microballoon dilution is also comprised.
2. the kit of detection expression of recombinant proteins situation according to claim 1, is characterized in that: also comprise luminescence-producing reaction plate, and preferably, described luminescence-producing reaction plate is the luminous check-out consoles in 96 holes.
3. use the kit of the detection expression of recombinant proteins situation as described in claim 1 ~ 2 to carry out the method detected, it is characterized in that: comprise following operation steps,
1) Escherichia coli after abduction delivering are carried out ultrasonication, namely obtain the broken bacterium liquid with His label destination protein;
2) by the acceptor ball solution of anti-for coupling His-tag antibody, add the mixing of luminescence-producing reaction plate with the broken bacterium liquid of His label destination protein, the Anti-TNF-α liquid solution of biotin labeled anti-destination protein, do 2 ~ 4 multiple holes, incubation 0.5 ~ 1.5 hour at 37 DEG C, preferably, incubation 1 hour; Negative control is set simultaneously, the operation of described negative control is that the ultrasonication bacterium liquid of the acceptor ball solution of anti-for coupling His-tag antibody, non-abduction delivering, the Anti-TNF-α liquid solution of biotin labeled anti-destination protein are added the mixing of luminescence-producing reaction plate, do 2 ~ 4 multiple holes, heated culture temperature and the time identical with hatching of the broken bacterium liquid with His label destination protein;
3) under lucifuge condition, the donor ball solution of Avidin mark is added to each hole, incubation 10 ~ 20min at 37 DEG C; Preferably, 15min, the donor ball solution concentration of Avidin mark is 20 mcg/ml;
4) reading, result judges; As the signal value of signal value >=2 negative control of bacterium liquid to be detected, be then determined with the great expression of recombinant protein; Preferably, by light stimulated luminescence detector sensed light signal under 615nm.
4. the kit detecting expression of recombinant proteins situation that uses according to claim 3 carries out the method detected, and it is characterized in that: step 1) in, with the OD of the broken bacterium liquid of His label destination protein
600nmbe 0.1 ~ 0.5.
5. the kit detecting expression of recombinant proteins situation that uses according to claim 3 carries out the method detected, it is characterized in that: described step 2) in, the acceptor ball solution of coupling anti-His-tag antibody, be 1:1:1 with the volume ratio of the broken bacterium liquid of His label destination protein, the Anti-TNF-α liquid solution of biotin labeled anti-destination protein.
6. the kit of the use detection expression of recombinant proteins situation according to claim 3 or 5 carries out the method detected, it is characterized in that: the acceptor ball solution of described coupling anti-His-tag antibody be the acceptor ball of the anti-His-tag antibody of coupling and acceptor dilution by volume 1:50 carry out allocating, preferably, acceptor dilution is Tris-HCl damping fluid, its pH is 8.0, and concentration is 0.1mol/L.
7. the kit that use according to claim 3 or 5 detects expression of recombinant proteins situation carries out the method detected, and it is characterized in that: the Anti-TNF-α liquid solution of described biotin labeled anti-destination protein be the polyclonal antibody of biotin labeled anti-destination protein and biotin antibody dilution by volume 1:2000 carry out allocating; Preferably, described biotin antibody dilution is PBS damping fluid, and its pH is 7.4, and concentration is 0.1mol/L.
8. the kit detecting expression of recombinant proteins situation that uses according to claim 3 carries out the method detected, it is characterized in that: step 3) in, the donor ball of the Avidin mark added to each hole is 1:7 with the volume ratio with the broken bacterium liquid of His label destination protein.
9. kit as claimed in claim 1 or 2 is detecting the application in expression of recombinant proteins.
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| CN107102050A (en) * | 2017-05-16 | 2017-08-29 | 肖玲君 | A kind of method for the expression for quantitatively determining the destination protein expressed in microorganism |
| CN109187484A (en) * | 2018-09-13 | 2019-01-11 | 广西师范大学 | A method of biotin is detected with compatible reaction regulation carbon dots catalysis SERS |
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Application publication date: 20151111 |