CN105624178B - It is a kind of based on the bimolecular fluorescence light segments complementary system of fluorescin iRFP and application - Google Patents
It is a kind of based on the bimolecular fluorescence light segments complementary system of fluorescin iRFP and application Download PDFInfo
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Abstract
The present invention is a kind of based on the bimolecular fluorescence light segments complementary system of fluorescin iRFP and application, using phytochrome fluorescin iRFP as template, not fluorescent nitrogen end and carbon teminal segment iRN97 and iRC98 are split as at 97-98, amino acid, when the two segments are respectively with the albumen of interaction to expression is blended, not fluorescent two segments can be furthered and be generated the fluorescence of iRFP；IRN97 and iRC98 blends expression with AIDS virus integrase IN and cell protein P75 respectively, pass through the fluorescence complementary of iRN97 and iRC98, the interaction of IN and p75 are studied in living cells, when the interaction between drug is able to suppress the protein-protein, iRN97 and iRC98 cannot then be furthered, to inhibit the recovery of fluorescence.The new system is a kind of evaluation system of drug that is simple, effectively, easily inhibiting protein-interacting.
Description
Technical field
The present invention relates to the technical fields for the drug evaluation for inhibiting protein-interacting, are more particularly to a kind of based on fluorescence
The bimolecular fluorescence light segments complementary system of albumen i RFP and application.
Background technique
Fluorescence light segments complementary system is a kind of using fluorescin as the fragment complementation system of material.The basic principle is that closing
Fluorescin is split into nitrogen end and carbon teminal segment by suitable site, and if this two segment not in the effect of interaction protein
Under, complementation that cannot be spontaneous restores fluorescence.If have the albumen of a Thermodynamic parameters respectively with the nitrogen end of the fluorescin of fractionation and
Carbon teminal segment blends, under the action of this Thermodynamic parameters albumen, the nitrogen end segment and carbon teminal piece of the fluorescin of fractionation
Section will be close to each other, to restore the conformation of complete fluorescin and issue specificity fluorescent.Fluorescence light segments complementary system energy
Enough specificity detect weak moment interaction, are a kind of easy, intuitive, sensitive sides for detecting protein-interacting
Method.
Antiviral drugs screening and appraisement system based on fluorescence light segments complementary system will provide simple, straight for drug development
Sight, the visual, drug screening of quantification and evaluation platform technology, provide important appraisement system in medicament research and development, thus
The development and diffusion for controlling epidemic situation, ensure the security of the lives and property of the people and aspect of safeguarding national security plays important work
With.
Currently, being directed to the forward position heat that particular target design and screening lead compound etc. are domestic and international medicament research and development research
Point.The suppression of the integrase of targeting HIV-1 and the LEDGF/p75 protein-interacting of the mankind is such as screened using virtual screening method
Preparation, to provide strategy (Hu GP et al.J Mol Model.2012 for treatment HIV-1；18:4995-5003).But
Most of these methods are cannot intuitively to show its effect inhibited, and the guide's chemical combination screened based on external research
Object needs further verify its activity on cellular level or living body level.Current screening and verification method also lacks very much.Thing
In reality, virus needs the interaction and viral or its important cause of many albumen in invasion cell processes or reproduction process
The interaction of cause of disease and host cell constituents, and it is enable to invade and survive.These protein-interactings infect thin
Some committed steps of born of the same parents are the major targets of antiviral drugs design and screening.Based between protein molecular interaction imaging and
Virus or its image analysing computer of key pathogenetic factor in the cell, will be provided for drug screening and evaluating drug effect most directly, really,
Visual research system provides important technical support for drug screening or evaluation.Certainly, the screening based on molecular image and
Appraisement system can also be combined further with high-flux medicaments sifting (HTS) or High content screening (HCS), certainly will be drug sieve
Choosing provides a kind of easy, feasible, real-time, visual screening and evaluation system.
So a kind of fluorescence light segments complementary system evaluated for inhibitor medicaments in living cells of this research and establishment, is used
In the drug of the evaluation inhibition protein-interacting in living cells level.The new system is based on distinguishing in same intracellular coexpression
The albumen pair that expression is blended with the nitrogen end segment iRN97 and carbon teminal segment iRC98 of iRFP, at inhibitor medicaments to be evaluated
Manage cell.By the fluorescence intensity for measuring intracellular iRFP, so that it may evaluate the inhibitor medicaments to albumen Thermodynamic parameters
Inhibit function.The system is a kind of flourescent sheet simply, effectively, conveniently, reliably evaluated for inhibitor medicaments in living cells
Section complementary system.
Summary of the invention
The purpose of the invention is to provide a kind of bimolecular fluorescence light segments complementary system based on fluorescin iRFP.
The system includes carrier piRN97 and piRC98, and wherein piRN97 carrier is the pcDNA3.1 for including sequence SEQ ID NO.2
Carrier, piRC98 carrier are the pcDNA3.1 carrier for including sequence SEQ ID NO.3.The system is a kind of simple, effective, side
Just, reliably inhibit the evaluation system of the drug of protein-interacting.
It is another object of the present invention to provide a kind of, and the bimolecular fluorescence light segments based on fluorescin iRFP are complementary
Systematic difference, the system can be used for the interaction of albumen, or the drug screening for inhibiting protein-interacting.
Mentality of designing of the invention is as follows:
With the phytochrome fluorescin iRFP (its sequence is shown in SEQ ID NO.1) from bacterium for material, choosing
Suitable fractionation site is taken, iRFP is split as two sections between amino acid 97 and 98, two is given expression to respectively and does not fluoresce
Nitrogen end and carbon teminal protein fragments iRN97 and iRC98, when albumen of the two segments respectively with a Thermodynamic parameters blends simultaneously
Coexpression is in same intracellular, two albumen drawings that fluorescing protein segment iRN97 and iRC98 can not interacted
Closely, the complete conformation of albumen iRFP is reverted to again and generates fluorescence；When drug be able to suppress it is mutual between the protein-protein
When effect, iRN97 and iRC98 cannot then be furthered, to inhibit the recovery of fluorescence.It is glimmering that success constructs the bimolecular based on iRFP
Light complementary system.Since this bimolecular fluorescence complementary system has the advantages that long wavelength, 37 degree of lower maturations, it can be just
The interaction between albumen is studied under normal physiological condition, which is that one kind simply, effectively, conveniently, reliably inhibits
The evaluation system of the drug of protein-interacting.
In order to achieve the above purpose, the present invention uses following technical measures:
A kind of bimolecular fluorescence light segments complementary system based on fluorescin iRFP, the system include carrier piRN97 and
piRC98。
The piRN97 carrier is the pcDNA3.1 carrier for including sequence SEQ ID NO.2；PiRC98 carrier is packet
PcDNA3.1 carrier containing sequence SEQ ID NO.3.
Specifically, piRN97 carrier the preparation method is as follows:
Nucleotide sequence shown in SEQ ID NO.2 (present invention or iRN97 gene) is utilized into double enzyme site EcoRV
And XhoI, iRN97 gene is inserted into the multiple cloning sites of pcDNA3.1 carrier to get carrier piRN97 (Fig. 1-A)
Specifically, piRC98 carrier the preparation method is as follows:
Using double enzyme site NheI and HindIII, by nucleotide sequence shown in SEQ ID NO.3 (present invention or
IRC98 gene) it is inserted into the multiple cloning sites of pcDNA3.1 carrier to get piRC98 (Fig. 1-B).
A kind of application of the bimolecular fluorescence light segments complementary system based on fluorescin iRFP, including for studying albumen
Interaction, or for studying the drug screening for inhibiting protein-interacting, detailed process is as follows:
It is a kind of based on the bimolecular fluorescence light segments complementary system of fluorescin iRFP research albumen interaction in
Using:
1) gene order of the bJun synthesized (is synthesized, reference literature Hu CD et al.Mol by the raw work in Shanghai
Cell.2002；9:789-98.), using double enzyme site NheI and HindIII, the more of bJun gene insertion piRN97 carrier
On cloning site, it is built into carrier pbJun-iRN97；
2) gene order of the bFos synthesized (is synthesized, reference literature Hu CD et al.Mol by the raw work in Shanghai
Cell.2002；9:789-98.), using double enzyme site KpnI and NotI, more grams of bFos gene insertion plasmid piRC98
On grand site, it is built into carrier piRC98-bFos；
3) it is intracellular to be transfected into Hela jointly by plasmid pbJun-iRN97 and piRC98-bFos, and 37 DEG C after culture 24 hours,
The albumen bJun-iRN97 and iRC98-bFos with bJun and bFos amalgamation and expression, the phase based on bJun and bFos are given expression to respectively
Interaction can further two protein fragments iRN97 and iRC98, so that it is restored original iRFP conformation, and can generate red
Fluorescence.And as control, although bFos the plasmid pbJun-iRN97 and piRC98-mbFos for being substituted for mbFos can be given expression to
Two protein fragments iRN97 and iRC98, but not generate fluorescence.Illustrate that the pUC pUC based on piRN97 and piRC98 can be with
For studying interactions between protein.
It is a kind of based on the bimolecular fluorescence light segments complementary system of fluorescin iRFP inhibit protein-interacting drug
Application in screening:
1) using the RNA of Hela cell as template, it is next step template by the cDNA that random primer amplifies, passes through PCR
Amplification obtains the gene order of host cell proteins LEDGF/p75, the upstream primer that PCR is used: 5 '-CGGCTAGCGCCAC
CATGACTCGCGATTTCAAACCTGGAGACC-3 ', downstream primer: 5 '-CCCAAGCTTGTTATCTAGT
P75 gene is inserted into pi RN97 using double enzyme site NheI and HindIII by GTAGAATCCTTCAGAGATATTTCAG-3 '
Obtain plasmid pp75-iRN97；
2) with plasmid pAD8 (Theodore TS et al.AIDS Res Hum Retroviruses.1996 Feb10；
12 (3): 191-4.) it is template, the gene order of AIDS virus integrase (IN), the upstream that PCR is used are obtained by PCR amplification
Primer: 5 '-GGGGTACCGAGCCACCCCCTCCTTTTTTGGATGGAATAGAT-3 ', downstream primer: 5 '-ATTTGCGGCCG
CTTAATCCTCATCCTGTCTACTTGC-3 ' is inserted into integrase IN gene using double enzyme site KpnI and NotI
PiRC98 obtains plasmid piRC98-IN.
3) it is intracellular to be transfected into 293T jointly by plasmid pp75-iRN97 and piRC98-IN, and 37 DEG C after culture 24 hours, respectively
Give expression to the albumen p75-iRN97 and iRC98-IN of amalgamation and expression, the interaction based on cell protein p75 and integrase IN,
Two protein fragments iRN97 and iRC98 can be furthered, so that it is restored original iRFP conformation, and red fluorescence can be generated.
4) plasmid transfection for expressing the albumen pair blended with iRN97 and iRC98 respectively is changed into liquid to mammalian cell
When inhibitor medicaments are added, by total incubation in 20-24 hours, by measuring the fluorescence intensity of iRFP, and compared with the control group
Compared with, so that it may Visual evaluation is carried out to inhibitor medicaments in living cells.
Compared with prior art, the invention has the characteristics that:
A, containing two plasmids piRN97 and piRC98 for needing cotransfection, the fluorescin of bacterial origin can be given expression to respectively
The nitrogen end segment iRN97 and carbon teminal segment iRC98 of iRF P；
B, contain ammonia benzyl mycin resistant gene, can be used for converting has the bacterium of the cotransfection plasmid on resistance culture base
Screening and culture；
C, it is inserted into interaction protein to be studied respectively in the corresponding multiple cloning sites of plasmid piRN97 and piRC98
Pair, it can be achieved that interaction protein Thermodynamic parameters to be studied research；
D, the plasmid transfection for expressing the albumen pair blended with iRN97 and iRC98 respectively is changed into liquid to mammalian cell
When inhibitor medicaments are added, by total incubation in 20-24 hours, by measuring the fluorescence intensity of iRFP, and compared with the control group
Compared with, so that it may Visual evaluation is carried out to inhibitor medicaments in living cells.
E, the system for when inhibitor medicaments evaluation, only need in living cells two plasmids of cotransfection for express respectively with
The albumen pair that iRFP nitrogen end segment iRN97 and carbon teminal segment iRC98 are blended separately adds an EGFP plasmid as internal reference, i.e.,
It can be achieved to screen inhibitor medicaments to be studied.Due to this bimolecular fluorescence complementary system can under 37 degree it is mature,
So the interaction between albumen can be studied under the normal physiological condition of cell.Simultaneously as glimmering based on phytochrome
The bimolecular fluorescence complementary system of photoprotein i RFP has the advantage of long wavelength, can study egg on living animal well
White interaction, therefore, this evaluation system may be applied to evaluate inhibitor medicaments on living animal.Due to iRFP and
The fluorescence detection of EGFP can directly detect, by the fluorescence signal for measuring iRFP without destroying cell under fluorescence microscope
Inhibitor medicaments can easily be evaluated to the inhibitory effect of protein-interacting.
F, this system and is usually considered based on the fluorescence complementary system for splitting and constructing between 97-98 to iRFP
It carries out splitting between the domain PAS and GAF of iRF P and be very different, the site 97-98 is not between two domains but exists
In deviate two domains a loop ring in, so most start selection split site when be easy to be ignored, meanwhile, this site
The fluorescence complementary efficiency for splitting and obtaining will be significantly higher than the complementary efficiency obtained between two domains.
Detailed description of the invention
Fig. 1-A is the building schematic diagram of carrier piRN97.
Fig. 1-B is the building schematic diagram of carrier piRC98.
Fig. 1-C is the statistical chart of the fluorescence complementary efficiency of different loci.
Fig. 2 interacts glimmering for the bimolecular fluorescence light segments complementary system research p75 and IN based on fluorescin iRFP
Light schematic diagram.
Fig. 3-A is that compound 6 (HIV-1 integrase inhibitor 2) verifies fluorescence light segments complementary system for living
The fluorogram of intracellular inhibitor medicaments evaluation feasibility；
Fig. 3-B is the corresponding relationship of compound 6 (HIV-1 integrase inhibitor 2) dosage and fluorescence intensity
Figure.
Fig. 4-A is fluorogram of the fluorescence light segments complementary system based on iRFP in living cells inner evaluation carbidopa.
Fig. 4-B is the corresponding relationship of carbidopa dosage and fluorescence intensity.
Specific embodiment
Technical solution described in the embodiment of the present invention is if not otherwise specified the conventional scheme of this field.
Embodiment 1:
A kind of building of the bimolecular fluorescence light segments complementary system based on the site fluorescin iRFP 97-98, building process
The following steps are included:
(1), it (shown in iRFP sequence SEQ ID NO.1, is synthesized by the raw work in Shanghai from plasmid pcDNA3.1-iRFP, passes through Nh
EI and HindIII restriction enzyme site is inserted into pcDNA3.1 and is prepared) on pass through PCR amplification (upstream primer: 5 '-GAAG
ATCTATGGCTGAAG GATCCGTC-3 ', downstream primer: 5 '-AAGGTACCCATCGTGAAGCCGACA GTG-3 ') it obtains carefully
The gene order of the nitrogen end segment iRN97 of the fluorescin iRFP in bacterium source, using double enzyme site BglII and KpnI,
IRN97 gene is inserted into the multiple cloning sites of pEGFP-C1 (being purchased from Clontech) carrier, is built into carrier pEGFP-iRN97；
(2), pass through PCR amplification (upstream primer: 5 '-GAAGATCTATGATGCGAAA from plasmid pcDNA3.1-iRFP
GGACG-3 ', downstream primer: 5 '-CCCAAGCTTCTCTTCCATCACGCCGATCTGCCAGG-3 ') obtain the glimmering of bacterial origin
The gene order of the carbon teminal segment iRC98 of photoprotein iRFP, using double enzyme site BglII and HindIII, iRC98 gene
It is inserted into the multiple cloning sites of pEGFP-N1 (being purchased from Clontech) carrier, is built into carrier pEGFP-iRC98；
(3) control group be with enzyme NheI and BglII cut off pEGFP-iRN97 in EGFP sequence, and with enzyme BamHI with
EGFP sequence in NotI excision pEGFP-iRC98 and the plasmid that constructs respectively, due to eliminating the interaction of EGFP, so
IRN97 segment cannot complementary generation specificity fluorescent with iRC98 segment.
(4), it is intracellular to be transfected into Hela jointly by plasmid pEGFP-iRN97 and pEGFP-iRC98, and 37 DEG C are cultivated 24 hours
Afterwards, the albumen EGFP-iRN97 and iRC98-EGFP with green fluorescent protein EGFP amalgamation and expression are given expression to respectively, are based on EGF P
The interaction of itself can further two protein fragments iRN97 and iRC98, it is made to restore original iRFP conformation, and energy
Generate red fluorescence.
And as control, although two protein fragments iRN97 can be given expression to by not merging EGFP plasmid piRN9 and piRC98
And iRC98, but not generate fluorescence.Illustrate to can be used for building based on two pUC pUCs of pEGFP-iRN97 and pEGFP-iRC98
Found novel bimolecular fluorescence light segments complementary system.
(5), it in addition chooses the site 122-123 between two domain PAS and GAF of iRFP and reports before this same
The site in the cross-domain region 120-121 between two domains constructs fluorescence light segments complementary system, construction method and above-mentioned building
Fluorescence complementary system based on the site 97-98 is identical.
Excitation channel selected by EGFP green fluorescence is 488nm, and excitation channel selected by iRFP red fluorescence is
640n m；The calculation of fluorescence complementary efficiency is the ratio of the complementary iRFP fluorescence intensity generated and the fluorescence intensity of EGFP,
The statistical result of complementary efficiency is as shown in Fig. 1-C.The site 97-98 fluorescence complementary efficiency obtained is the 3.1 of the site 122-123
Times, it is 1.8 times of the site 120-121.
Embodiment 2:
A kind of bimolecular fluorescence light segments complementary system based on fluorescin iRFP, building process the following steps are included:
1) pass through PCR amplification (upstream primer: 5 '-GGGATATCGAGCCACCCCCTC from plasmid pEGFP-iRN97
CTATGGCTGAAGGATCCGTC-3 ', downstream primer: 5 '-CCGCTCGAGTTACATCGTGAAGCCGA CAG-3 ') it obtains carefully
The gene order (shown in SEQ ID NO.2) of the nitrogen end segment iRN97 of the fluorescin iRFP in bacterium source, utilizes double enzyme site
EcoRV and XhoI is inserted into iRN97 gene in the multiple cloning sites of pcDNA3.1 carrier, i.e. plasmid piRN97 (Fig. 1-A)；
2) pass through PCR amplification (upstream primer: 5 '-CGGCTAGCGCCACCATGCGA from plasmid pEGFP-iRC98
AAGGACGCAGGCTTCATC-3 ', downstream primer: 5 '-CCCAAGCTTCTCTTCCATCACGCCGATC TGCC-3 ') it obtains carefully
The gene order (shown in SEQ ID NO.3) of the carbon teminal segment iRC98 of the fluorescin iRFP in bacterium source, utilizes double enzyme site
NheI and HindIII is inserted into iRC98 gene in the multiple cloning sites of pcDNA3.1 carrier, i.e. plasmid piRC98 (Fig. 1-B)；
Embodiment 3:
It is a kind of based on the bimolecular fluorescence light segments complementary system of fluorescin iRFP research albumen interaction in
Using application process is as follows:
1) gene order of the bJun synthesized (is synthesized, reference literature Hu CD et al.Mol by the raw work in Shanghai
Cell.2002；9:789-98.), using double enzyme site NheI and HindIII, the more of bJun gene insertion piRN97 carrier
On cloning site, it is built into carrier pbJun-iRN97；
2) gene order of the bFos synthesized (is synthesized, reference literature Hu CD et al.Mol by the raw work in Shanghai
Cell.2002；9:789-98.), using double enzyme site KpnI and NotI, more grams of bFos gene insertion plasmid piRC98
On grand site, it is built into carrier piRC98-bFos；
3) control group is to use upstream primer 5 '-GGGGTACCGAGCCACCCCCTCCTATGGGTCGTGCGCAGTC-3 '
Gone out using bFos as template amplification with downstream primer 5 '-ATTTGCGGCCGCTTAACCCAGGTCGTTCGGGATTTTGC-3 '
MbFos replaces corresponding bFos and the plasmid that constructs, and mbFos and bJun lose interaction to each other.
4) it is intracellular to be transfected into Hela jointly by plasmid pbJun-iRN97 and piRC98-bFos, and 37 DEG C after culture 24 hours,
The albumen bJun-iRN97 and iRC98-bFos with bJun and bFos amalgamation and expression, the phase based on bJun and bFos are given expression to respectively
Interaction can further two protein fragments iRN97 and iRC98, so that it is restored original iRFP conformation, and can generate red
Fluorescence.And as control, although bFos the plasmid pbJun-iRN97 and piRC98-mbFos for being substituted for mbFos can be given expression to
Two protein fragments iRN97 and iRC98, but not generate fluorescence.Illustrate that the pUC pUC based on piRN97 and piRC98 can be with
For establishing novel bimolecular fluorescence light segments complementary system.
Embodiment 4:
It is a kind of based on the bimolecular fluorescence light segments complementary system of fluorescin iRFP inhibit protein-interacting drug
Application in screening,
Albumen is chosen to p75 and IN, the interaction of p75 and IN are had studied with bimolecular complementary system of the invention, it
Afterwards, the inhibitor medicaments compound 6 of p75 and IN interaction can be inhibited to use to verify this system in living cells level known to selection
In the reliability that inhibitor medicaments in living cells are evaluated.Finally, protein-interacting inhibitor carbidopa to be evaluated is chosen,
The addition when transfecting 5h and changing liquid passes through the fluorescence intensity of iRFP under the conditions of measurement different disposal, while cotransfection green fluorescence egg
White EGFP evaluates influence of the inhibitor to cell transfecting efficiency as internal reference, finds to block eventually by compared with the control group
Than the efficiency that DOPA will not influence cell transfecting, but by it is a kind of it is dose-dependent in a manner of inhibit phase between p75 and IN
Interaction, in the living cells fluorescence light segments complementary system of inhibitor medicaments evaluation for inhibitor medicaments evaluation in living cells,
Evaluation method is easy, easy to operate, only need to transfect two plasmids and be added inhibitor medicaments, can be real by fluorescent microscopic imaging
Evaluation in existing living cells to inhibitor medicaments.
Its application process is as follows:
1) using the RNA of Hela cell as template, it is next step template by the cDNA that random primer amplifies, passes through P CR
Amplification obtains the gene order of host cell proteins LEDGF/p75, the upstream primer that PCR is used: 5 '-CGGCTAGC GCCACC
ATGACTCGCGATTTCAAACCTGGAGACC-3 ', downstream primer: 5 '-CCCAAGCTTGTTA
TCTAGTGTAGAATCCTTCAGAGATATTTCAG-3 ' is inserted into p75 gene using double enzyme site NheI and HindIII
PiRN97 obtains plasmid pp75-iRN97；
2) with plasmid pAD8 (Theodore TS et al.AIDS Res Hum Retroviruses.1996 Feb10；
12 (3): 191-4.) it is template, the gene order of AIDS virus integrase (IN), the upstream that PCR is used are obtained by PCR amplification
Primer: 5 '-GGGGTACCGAGCCACCCCCTCCTTTTTTGGATGGAATAGAT-3 ', downstream primer: 5 '-ATTTGCGGCCG
CTTAATCCTCATCCTGTCTACTTGC-3 ' is inserted into integrase IN gene using double enzyme site Kpn I and NotI
PiRC98 obtains plasmid piRC98-IN.
3) it is intracellular to be transfected into 293T jointly by plasmid pp75-iRN97 and piRC98-IN, and 37 DEG C after culture 24 hours, respectively
Give expression to the albumen p75-iRN97 and iRC98-IN of amalgamation and expression, the interaction based on cell protein p75 and integrase IN,
Two protein fragments iRN97 and iRC98 can be furthered, so that it is restored original iRFP conformation, and red fluorescence can be generated.
4) dense by series by plasmid pp75-iRN97 and piRC98-IN cotransfection to mammalian cell 293T cell
The compound 6 (HIV-1 integrase inhibitor 2) of degree handles 293T cell, passes through the fluorescence intensity of measurement iRFP
And with the DMSO of equivalent processing control group compared with, find compound 6 (HIV-1 integrase inhibitor 2) with
A kind of dose-dependent mode inhibits the interaction (Fig. 3-A) of p75 and IN, and the corresponding relationship of dosage and fluorescence intensity is as schemed
(specific value is as shown in the table) shown in 3-B illustrates that constructed fluorescence light segments complementary system can be used for inhibitor medicaments
Evaluation.
| The concentration (μM) of compound 6 | 0 | 1 | 2 | 5 | 25 | 125 |
| Fluorescence intensity (a.u.) | 1 | 0.947 | 0.844 | 0.746 | 0.626 | 0.472 |
5) plasmid pp75-iRN97 and piRC98-IN cotransfection are passed through to mammalian cell 293T cell and are after
The carbidopa of column concentration handles 293T cell, by measuring the fluorescence intensity of iRFP and handling with the dilute hydrochloric acid of equivalent
Control group compares, discovery carbidopa be also by it is a kind of it is dose-dependent in a manner of inhibit interaction (Fig. 4-of p75 and IN
A), dosage and the corresponding relationship of fluorescence intensity as shown in Fig. 4-B (specific value is as shown in the table) illustrate carbidopa living thin
Born of the same parents' level can also inhibit the interaction of p75 and IN.
| The concentration (μM) of carbidopa | 0 | 6.5 | 16.25 | 32.5 | 65 | 97.5 | 130 | 162.5 |
| Fluorescence intensity (a.u.) | 1 | 0.956 | 0.910 | 0.795 | 0.714 | 0.651 | 0.514 | 0.404 |
SEQUENCE LISTING
<110>Wuhan Virology Institute,Chinan academy of Sciences
<120>a kind of based on the bimolecular fluorescence light segments complementary system of fluorescin iRFP and application
<130>a kind of based on the bimolecular fluorescence light segments complementary system of fluorescin iRFP and application
<160> 3
<170> PatentIn version 3.1
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<211> 951
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atggctgaag gatccgtcgc caggcagcct gacctcttga cctgcgacga tgagccgatc 60
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atcgttgccg gcagcgacaa ccttcccgaa ctcaccggac tggcgatcgg cgccctgatc 180
ggccgctctg cggccgatgt cttcgactcg gagacgcaca accgtctgac gatcgccttg 240
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gcaggcttca tcggctcctg gcatcgccat gatcagctca tcttcctcga gctcgagcct 360
ccccagcggg acgtcgccga gccgcaggcg ttcttccgcc gcaccaacag cgccatccgc 420
cgcctgcagg ccgccgaaac cttggaaagc gcctgcgccg ccgcggcgca agaggtgcgg 480
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attgatctta gcttcgccat cctgcgcagc gtctcgcccg tccatctgga attcatgcgc 780
aacataggca tgcacggcac gatgtcgatc tcgattttgc gcggcgagcg actgtgggga 840
ttgatcgttt gccatcaccg aacgccgtac tacgtcgatc tcgatggccg ccaagcctgc 900
gagctagtcg cccaggttct ggcctggcag atcggcgtga tggaagagtg a 951
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ggccgctctg cggccgatgt cttcgactcg gagacgcaca accgtctgac gatcgccttg 240
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gccatccgcc gcctgcaggc cgccgaaacc ttggaaagcg cctgcgccgc cgcggcgcaa 180
gaggtgcgga agattaccgg cttcgatcgg gtgatgatct atcgcttcgc ctccgacttc 240
agcggcgaag tgatcgcaga ggatcggtgc gccgaggtcg agtcaaaact aggcctgcac 300
tatcctgcct caaccgtgcc ggcgcaggcc cgtcggctct ataccatcaa cccggtacgg 360
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gggcggccga ttgatcttag cttcgccatc ctgcgcagcg tctcgcccgt ccatctggaa 480
ttcatgcgca acataggcat gcacggcacg atgtcgatct cgattttgcg cggcgagcga 540
ctgtggggat tgatcgtttg ccatcaccga acgccgtact acgtcgatct cgatggccgc 600
caagcctgcg agctagtcgc ccaggttctg gcctggcaga tcggcgtgat ggaagag 657
Claims (3)
1. a kind of bimolecular fluorescence light segments complementary system based on fluorescin iRFP, the system include carrier piRN97 and
piRC98；The piRN97 carrier is the pcDNA3.1 carrier for including sequence SEQ ID NO.2；PiRC98 carrier be comprising
There is the pcDNA3.1 carrier of sequence SEQ ID NO.3；
The preparation method of the carrier piRN97, specific as follows:
Nucleotide sequence shown in SEQ ID NO.2 is utilized into double enzyme site EcoRV and XhoI, iRN97 gene is inserted into pc
To get carrier piRN97 in the multiple cloning sites of DNA3.1 carrier；
The preparation method of the carrier piRC98, specific as follows:
Using double enzyme site NheI and HindIII, nucleotide sequence shown in SEQ ID NO.3 is inserted into pcDNA3.1 and is carried
To get piRC98 in the multiple cloning sites of body.
2. application of the system described in claim 1 in the interaction of research albumen.
3. application of the system described in claim 1 in the drug screening for inhibiting protein-interacting.
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| CN110128546B (en) * | 2019-04-28 | 2022-05-17 | 河北科技大学 | Fusion protein for RNA tracing and application thereof |
| CN112592936B (en) * | 2020-11-09 | 2023-03-31 | 中国科学院深圳先进技术研究院 | Tandem fragment fluorescence complementary system and construction method and application thereof |
| CN113667694B (en) * | 2021-07-30 | 2023-07-28 | 中国科学院深圳先进技术研究院 | Bimolecular fluorescence complementary system based on photopigment protein miRFP670nano |
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