CN103290091B - A kind of Protein interaction detection method of low false positive rate - Google Patents
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Abstract
The present invention is a kind of Protein interaction detection method of low false positive rate, relates to a kind of detection system, belongs to field of molecular biotechnology.The present invention utilizes the bimolecular fluorescence complementary technology based on fluorescin, be building up in a double expression plasmid carrier system by usually needing two BiFC detection system based on single expression plasmid carrier, obviously can reduce the false positive rate of BiFC detection method in protein-protein interaction research, and can quantitative analysis be realized.The present invention also can be used for the screening study of the agnoprotein matter-protein interaction based on gene library, and the protein-protein interaction in living animal detects.
Description
Technical field
The invention belongs to technical field of molecular biology, particularly a kind of Protein interaction detection method of low false positive rate.
Background technology
Cell itself is the elementary cell being formed an orderly vital movement by different biological molecules interphase interaction.The generation of the great vital movement of cell and adjustment are interacted by (protein-protein, protein-nucleic acid etc.) between biomacromolecule and realize.Protein-protein interaction (protein-protein interaction, hereinafter referred to as " PPI ") not only control genetic transcription, cell fission and cell proliferation, go back the signal transduction in mediated cell vital movement process, oncogenic transformation and adjustment etc., therefore Study on Protein-protein interaction has important life science meaning simultaneously.
At present, the technology of the PPI imaging of exact space location in viable cell can be realized for mammalian cell, only has FRET (fluorescence resonance energy transfer) (Fluorescence Resonance Energy Transfer, FRET) technology and bimolecular fluorescence complementary (Bimolecular Fluorescence Complementation, BiFC) technology.Based on the BiFC technology of fluorescin, refer to and utilize gene clone technology that complete fluorescin is cut formation two non-blooming fragments in appropriate site, and make these two fragments form fusion rotein with two target proteins respectively, the space length of these two fluorescence light segments that further by the interaction between two target proteins, thus be again folded to form structural integrity and there is the fusion rotein of fluorescent characteristic.Compared with FRET technology, the performance requriements of bimolecular fluorescence complementary technology to plant and instrument is lower, experimental result is more directly perceived, subsequent disposal is simple, detection sensitivity is higher, especially in the detection of weak PPI, have advantage.But existing BiFC technology, all along with higher false positive rate, also can produce very high background signal in the detection, these shortcomings the interaction of the method between agnoprotein matter is detected and PPI quantitative examination in be restricted.Therefore, develop the BiFC system of low false positive rate, greatly will promote that intravital protein function is studied.In addition, the BiFC technology based on multiple different fluorescin is utilized can to realize the detection of multipair protein interaction in viable cell easily.Usually, when carrying out BiFC detection for a pair PPI, need to contaminate two kinds of plasmids to transit cell simultaneously.If carry out BiFC detection for three couples of PPI simultaneously, then need to contaminate 6 kinds of plasmids to transit cell simultaneously.In obvious many plasmid transfections process, heterogeneic expression ratio is difficult to control.This problem have impact on the application of BiFC technology in multipair protein interaction detects to a great extent.
Summary of the invention
The object of the invention is for solving the problem and providing a kind of Protein interaction detection method of low false positive rate.Utilize the method while detection protein interaction, the false positive that can reduce BiFC significantly produces.
The technical solution adopted in the present invention is:
A Protein interaction detection method for low false positive rate, comprises the following steps:
1) fluorescin is cut into two protein sequence fragments at specific site, the N end of fluorescin and C end called after FPN and FPC respectively;
2) DNA sequence dna of target protein A to be measured and FPN gene order are inserted into simultaneously the downstream of a promotor in dual-expression vector, another testing protein B and another promotor downstream of simultaneously inserting with FPC gene order in dual-expression vector, thus construct the bimolecular fluorescence complementary system based on dual-expression vector;
3) by the bimolecular fluorescence complementary system transfectional cell simultaneously containing target protein A and B to be measured, cultivate after 16 ~ 24 hours, use before and after fluorescence microscope source stimulating whether there is fluorescence; Interact if two target proteins exist, then the N end of fluorescin and C end close, thus be spontaneously reassembled as complete fluorescin, generation fluorescent signal; If not or only have extremely week fluorescent, prove between two target proteins without interacting.
Preferably, described fluorescin is DsRed mLumin far away, yellow fluorescence protein mVenus and cyan fluorescent protein mCerulean.
Preferably, described dual-expression vector is pBudCE4.1, pIRES2 and pVITR03.
Preferably, described step 3) in, interact when two target proteins exist, the N end of fluorescin and C hold close distance range to be 10nm ~ 20nm, thus are spontaneously reassembled as complete fluorescin, generation fluorescent signal.
Further, when described fluorescin is DsRed mLumin far away, specific site is No. 155 Serine.
Further, when described fluorescin is yellow fluorescence protein mVenus, specific site is No. 155 Serines and No. 173 Serines.
Further, when described fluorescin is cyan fluorescent protein mCerulean, specific site is No. 155 Serines and No. 173 Serines.
The present invention has the following advantages:
Present method detects the interaction of protein under can meeting viable cell condition, based on the dual-expression vector system of fluorescin, compared with BiFC system (the common transfections of at least two carrier molecules) conventional at present, operate easier, the carrier amount of transfection is more easy to control, the expression amount of often pair of fusion rotein is more stable, can be used for the quantitative analysis of protein interaction; The method significantly reduces the false-positive generation of BiFC simultaneously, expands the range of application of BiFC technology, makes it to be applicable to from gene library, filter out unknown protein-protein interaction quickly and efficiently.
Accompanying drawing explanation
Below in conjunction with drawings and embodiments, the present invention is further detailed explanation.
Fig. 1 is the dual-expression vector plasmid map of mLumin-BiFC system;
Fig. 2 is that the transfection of COS-7 cell difference pBud-Ln-bFos-Lc-bJun, pBud-Ln-b △ Fos-Lc-bJun, pBud-Ln-bFos-bJun-Lc and pBud-Ln-b △ Fos-bJun-Lc is in 37 DEG C of cultivation fluorescence imaging figure after 24 hours;
Fig. 3 is that the transfection of COS-7 cell difference pBud-Ln-bFos-Lc-bJun, pBud-Ln-b △ Fos-Lc-bJun, pBud-Ln-bFos-bJun-Lc and pBud-Ln-b △ Fos-bJun-Lc is in the data statistic analysis histogram of 37 DEG C of cultivations after 24 hours;
Fig. 4 is that the transfection of COS-7 cell difference pBud, pBud-Ln-b △ Fos-bJun-Lc and pBud-Ln-bFos-bJun-Lc is in 37 DEG C of cultivation flow cytometry analysis result figure after 24 hours;
Fig. 5 is that the transfection of COS-7 cell difference pBud, pBud-Ln-b △ Fos-bJun-Lc and pBud-Ln-bFos-bJun-Lc is in the data statistic analysis histogram of 37 DEG C of cultivations after 24 hours;
Fig. 6 is that the transfection of COS-7 cell difference bJun-Vn/bFos-Vc, bJun-Vn/ △ bFos, pBud-Vn-bFos-Vc-bJun and pBud-Vn-b △ Fos-Vc-bJun is in 37 DEG C of cultivation fluorescence imaging figure after 24 hours;
Fig. 7 is that the transfection of COS-7 cell difference bJun-Vn/bFos-Vc, bJun-Vn/ △ bFos, pBud-Vn-bFos-Vc-bJun and pBud-Vn-b △ Fos-Vc-bJun is in the data statistic analysis histogram of 37 DEG C of cultivations after 24 hours;
Fig. 8 is that the transfection of COS-7 cell difference pBud-Ln-RBD-Lc-bKRas, pBud-Ln-RBD-Lc-KRas12v, pBud-Ln-RBD-Lc-KRas185S are in 37 DEG C of cultivation fluorescence imaging figure after 24 hours;
Fig. 9 is that the transfection of COS-7 cell difference pBud-Ln-RBD-Lc-bKRas, pBud-Ln-RBD-Lc-KRas12v, pBud-Ln-RBD-Lc-KRas185S are in the data statistic analysis histogram of 37 DEG C of cultivations after 24 hours;
Figure 10 is that the transfection of COS-7 cell difference pBud-Ln-RBD-Lc-bKRas, pBud-Ln-RBD-Lc-KRas12v, pBud-Ln-RBD-Lc-KRas185S are in the laser co-focusing fluorescence imaging figure of the individual cells of 37 DEG C of cultivations after 24 hours;
Figure 11 is that the transfection of COS-7 cell difference pBud-Ln-Grb2-Lc-bKRas, pBud-Ln-Grb2-Lc-KRas12v, pBud-Ln-Grb2-Lc-KRas185S are in 37 DEG C of cultivation fluorescence imaging figure after 24 hours;
Figure 12 is that the transfection of COS-7 cell difference pBud-Ln-Grb2-Lc-bKRas, pBud-Ln-Grb2-Lc-KRas12v, pBud-Ln-Grb2-Lc-KRas185S are in the data statistic analysis histogram of 37 DEG C of cultivations after 24 hours;
Figure 13 is that the transfection of COS-7 cell difference pBud-Ln-Grb2-Lc-bKRas, pBud-Ln-Grb2-Lc-KRas12v, pBud-Ln-Grb2-Lc-KRas185S are in the laser co-focusing fluorescence imaging figure of the individual cells of 37 DEG C of cultivations after 24 hours.
Embodiment
The present invention is a kind of detection method of protein interaction, and based on red fluorescent protein mutant far away and yellow fluorescence protein, and the key of this detection method is, choosing of fluorescin and choosing of mammalian cell dual-expression vector.That select in this embodiment is DsRed mLumin far away and yellow fluorescence protein mVenus, and dual-expression vector uses carrier for expression of eukaryon pBudCE4.1.PBudCE4.1 in embodiment contains two strong promoter CMV and EF-1 α, C-terminal and bJun that far red/yellow fluorescence protein fragment is inserted in the N-terminal of far red/yellow fluorescence protein fragment and bFos, EF-1 α promotor downstream is inserted respectively in the downstream of CMV promoter.Make fluorescin fragment and bFos/bJun can amalgamation and expression, mediate under the interaction of bFos and bJun two fluorescin fragment complementations formed one complete can be luminous fluorescin.In principle, also can substitute this two promotors with other strong promoter, but should ensure that two promotors ability that promotor gene is expressed in identical carrier is suitable.
Fig. 1 is the dual-expression vector plasmid map of mLumin-BiFC system.It is complementary that two fragments (mLumin-155C and mLumin-155N) that DsRed mLumin the 155th amino acids far away is divided into mediate fluorescin by PPI, formation can be luminous complete fluorescin mLumin, thus the PPI optical imagery that can realize mLumin-BiFC probe detects.When building plasmid, pBud CE4.1 dual-expression vector used contains CMV and EF-1 α two, and independently promotor and Zeocin screen resistance.By restriction endonuclease Sal I/BamH I and NotI/Bgl II, fluorescence light segments mLumin-155N-bFos and mLumin-155C-bJun is inserted into CMV and EF-1 α promotor downstream respectively, makes it can simultaneously expressed fusion protein independently.Can via the interaction between bFos and bJun under 37 DEG C of conditions, make fluorescin fragment close to each other and form complete fluorescin that can be luminous, producing fluorescent signal.Other probe involved in the present invention also can use other restriction enzyme site in pBud CE4.1 vector multiple cloning site to carry out similar molecular cloning protocols, also can replacing bFos/bJun, being configured to the similar mLumin-BiFC probe for detecting other PPI.
Fig. 2 and Fig. 3 is the fluorogram of DsRed mutant mLumin-BiFC system far away after transfection COS-7 cell based on dual-expression vector.As can be seen from Figure 2, in this individual system, in positive and negative group, the cell number of express fluorescent protein has huge difference.In negative control group, almost can't see the cell sending red fluorescence, Fig. 3 also illustrates that the mLumin-BiFC system based on dual-expression vector creates difference significantly in the positive with negative group, reduces false-positive generation.
Fig. 4 and Fig. 5 is the flow cytometry of DsRed mutant mLumin-BiFC system far away after transfection COS-7 cell based on dual-expression vector.Result shows can reach 37.65 times based on the mLumin-BiFC fluorescent signal positive of dual-expression vector with feminine gender ratio.And the difference between negative group and blank group is also very little.This illustrates that the mLumin-BiFC system based on dual-expression vector can reduce false positive results greatly, and positive group has very strong fluorescence complementary efficiency simultaneously.
Fig. 6 to Fig. 7 is building up to (Vn-bFos/ Δ bFos+Vc-bJun) in two expression vectors respectively based on the BiFC system of yellow fluorescence protein mutant mVenus and is implemented in the plasmid of (Vn-bFos/ Δ bFos-Vc-bJun) in dual-expression vector, the fluorogram after transfection COS-7 cell.As can be seen from the figure, the BiFC system be implemented in two strongly expressed carriers the most frequently used at present, negative control still has a large amount of cell to produce fluorescent signal.And in dual-expression vector, the cell quantity and the fluorescence intensity that produce fluorescent signal in negative control group all significantly decrease.
Previous work of the present invention is the structure of BiFC intermediate carrier.
Its concrete steps are:
BiFC intermediate carrier pBud-Vn-Vc and pBud-Ln-Lc plasmid construction
Mlumin encoding sequence 1-155 (Ln), 156-234 (Lc) cut rear Hind III/Sal I site and Bgl II/Mlu I site of inserting pBudCE4.1 respectively by pcr amplification enzyme;
Building process primer sequence used is
Embodiment 1
This embodiment is the detection method utilizing the mLumin-BiFC system based on dual-expression vector to set up a kind of protein interaction of low false positive rate.Based on this object, held by the N of bFos and mLumin protein sequence and be connected (hereinafter referred to as LN), the C end of bJun and mLumin protein sequence is connected (hereinafter referred to as LC).
With reference to the BiFC molecular probe structure iron that Fig. 1, Fig. 1 are based on mLumin155 site, be made up of with negative group mLumin-Ln55-Δ bFos/mLumin-Lc155-bJun the positive group mLumin-Ln155-bFos/mLumin-Lc155-bJun of probe.Wherein Δ bFos is the bFos after deletion mutantion, can not interact under physiological condition with bJun, does not thus produce BiFC fluorescent signal.
Its concrete steps are: first using gene engineering technique builds pBud-Ln-Lc, and plasmid can express Ln and Lc respectively in mammalian cell.Application round pcr increases bFos (or Δ bFos) clone inserts the Ln downstream in pBud-Ln-Lc respectively; Amplification bJun clone insert the Lc upstream of pBud-Ln-Lc or downstream, and (bJun and bFos can interact, bJun and Δ bFos can not interact), namely four groups of plasmid vectors are built, be respectively pBud-Ln-Fos-Lc-Jun, pBud-Ln-Fos-Jun-Lc, pBud-Ln-Δ Fos-Lc-Jun and pBud-Ln-Δ Fos-Jun-Lc, thus obtain the plasmid can expressing BiFC molecular probe.Namely complete BiFC probe involved in the present invention to build.
Fig. 2 and Fig. 3 detects fluorogram in application and BiFC Efficiency Statistics result based on the dual-expression vector BiFC probe in mLumin155 site PPI in cell.As figure shows, respectively by after pBud-Ln-Fos-Lc-Jun and pBud-Ln-Fos-Jun-Lc transfection COS-7 cell, (spectral filter parameter 520-550 under common fluorescent microscope, DM565,580LP, Olympus), observe and find that there is very strong red fluorescent, and pBud-Ln-Δ bFos-Lc-Jun and pBud-Ln-Δ bFos-Jun-Lc probe almost do not detect red fluorescent.This illustrates that the mLumin-BiFC probe based on dual-expression vector has obvious yin and yang attribute contrast gradient, namely proves that the present invention can reduce false-positive generation, can be applied to the research of viable cell internal protein-protein interaction more easily.
Fig. 4 and Fig. 5 is the FCM analysis result of the mLumin-BiFC system based on dual-expression vector.In order to verify that the present invention can reduce the generation of false positive results further, have detected the far away red fluorescent signal in Yin/Yang test group after 10000 cell fluorescence complementations respectively by flow cytometry.Contrast the fluorescent signal per-cent of negative experimental group and blank group, more determine that the present invention can lower the fluorescent signal of negative experimental group.Show the present invention with the comparing result of positive test group effectively to reduce false positive and produce.
Fig. 6 and Fig. 7 be based on the mVenus-BiFC system transfections COS-7 cell of dual-expression vector after fluorogram and analysis of statistical results.As shown in the figure, by the BiFC system of contrast use two expression vectors, find that the mVenus-BiFC system based on dual-expression vector also can reduce false positive results.
Embodiment 2
Raf1 is the main effects albumen of the important composition molecule KRas of Cell signal transduction pathway, in growth, grows and plays an important role in cancer generation.The Ras binding domain RBD of Kras-Raf1 albumen is taken as the special probe of Ras activity in much research.The KRas of GTP combining form has stronger avidity than KRas and the RBD that GDP combines, and the avidity between Ras and the Raf1 that GDP combines is not enough to participate in intracellular signaling because of too weak.Therefore, the Heterodimerization of RBD and KRas can be used as the good protein interaction model of PPI detection method in development viable cell.
Fig. 8 to Figure 10 is the fluorogram after pBud-Ln-RBD-Lc-KRas, pBud-Ln-RBD-Lc-KRas12v and pBud-Ln-RBD-Lc-KrasC185S transfection COS-7 cell.RBD and KRas or KRas mutant are inserted in pBud-Ln-Lc carrier, constructs pBud-Ln-RBD-Lc-KRas, pBud-Ln-RBD-Lc-KRas12v and pBud-Ln-RBD-Lc-KrasC185S tri-groups of probes.Respectively after transfection COS-7 cell, under 37 DEG C of conditions, cultivate 24h.Fluorescence microscope finds to create red fluorescence on cytolemma or in full cell, shows that the interaction of the Kras of RBD and different structure occurs in the different spatial of cell.This result conforms to prior document report.And RBD and enhancement type KRas12v interacts, the red fluorescent produced obviously is better than natural type KRas.Visible genotype probe biomolecule provided by the present invention can detect the interaction of protein delicately in mammalian cell.
Embodiment 3
Grb2 is the important adaptin in cell-signaling pathways, can from different signal path downstream egg white hair looks mutual effects by Grb2, KRas.The activation of KRas completes mainly through itself and the effect of Grb2-SOS1 protein complexes.
Segment Grb2 and KRas or its mutant clone will be merged to pBud-Ln-Lc, construct pBud-Ln-Grb2-Lc-KRas, pBud-Ln-Grb2-Lc-KRas12v and pBud-Ln-Grb2-Lc-KRas185S.After plasmid difference transfection COS-7 cell, cultivate under 37 DEG C of conditions after 24 hours and carry out imaging.With reference to Figure 11 to Figure 13, the red fluorescence of cell on cytolemma that pBud-Ln-Grb2-Lc-KRas12v expresses obviously is better than pBud-Ln-Grb2-Lc-KRas, obvious fluorescence is then can't see in pBud-Ln-Grb2-Lc-KRas185S, illustrate that BiFC probe can be applied in the detection of the complex body formed more than two albumen, the activation also demonstrating KRas enhances the keying action between KRas and Grb2.
It should be noted last that, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (7)
1. a Protein interaction detection method for low false positive rate, is characterized in that, comprises the following steps:
1) fluorescin is cut into two protein sequence fragments at specific site, the N end of fluorescin and C end called after FPN and FPC respectively;
2) DNA sequence dna of target protein A to be measured and FPN gene order are inserted into simultaneously the downstream of a promotor in dual-expression vector, another testing protein B and another promotor downstream of simultaneously inserting with FPC gene order in dual-expression vector, thus construct the bimolecular fluorescence complementary system based on dual-expression vector; The ability that promotor gene is expressed in identical carrier of two promotors in described dual-expression vector is suitable;
3) by the bimolecular fluorescence complementary system transfectional cell simultaneously containing target protein A and B to be measured, cultivate after 16 ~ 24 hours, use before and after fluorescence microscope source stimulating whether there is fluorescence; Interact if two target proteins exist, then the N end of fluorescin and C end close, thus be spontaneously reassembled as complete fluorescin, generation fluorescent signal; If not or only have extremely week fluorescent, prove between two target proteins without interacting.
2. the Protein interaction detection method of low false positive rate according to claim 1, is characterized in that, described fluorescin is DsRed mLumin far away, yellow fluorescence protein mVenus and cyan fluorescent protein mCerulean.
3. the Protein interaction detection method of low false positive rate according to claim 1, is characterized in that, described dual-expression vector is pBudCE4.1, pIRES2 and pVITR03.
4. the Protein interaction detection method of the low false positive rate according to any one of claims 1 to 3, it is characterized in that, described step 3) in, interact when two target proteins exist, the N end of fluorescin and C hold close distance range to be 10nm ~ 20nm, thus be spontaneously reassembled as complete fluorescin, produce fluorescent signal.
5. the Protein interaction detection method of the low false positive rate according to any one of claims 1 to 3, is characterized in that, when described fluorescin is DsRed mLumin far away, specific site is No. 155 Serine.
6. the Protein interaction detection method of the low false positive rate according to any one of claims 1 to 3, is characterized in that, when described fluorescin is yellow fluorescence protein mVenus, specific site is No. 155 Serines and No. 173 Serines.
7. the Protein interaction detection method of the low false positive rate according to any one of claims 1 to 3, is characterized in that, when described fluorescin is cyan fluorescent protein mCerulean, specific site is No. 155 Serines and No. 173 Serines.
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