CN102127561B - Recombinant fluorescent dual-hybrid yeast and detection method and applications thereof - Google Patents
Recombinant fluorescent dual-hybrid yeast and detection method and applications thereof Download PDFInfo
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Abstract
The invention discloses recombinant fluorescent dual-hybrid yeast and a detection method and the applications thereof. The recombinant fluorescent yeast is a recombinant microzyme obtained by transferring a fusion gene into a microzyme Y187; and the fusion gene is formed by sequentially connecting DNA (Deoxyribonucleic Acid) fragments shown by the 9th bit to the 1030th bit in a sequence (5) in a sequence table, a reporter gene, a terminator, a selection marker protein expression cassette and DNA fragments shown by the 4336th bit to the 5226th bit in the sequence (5) in the sequence table. The recombinant fluorescent yeast provided by the invention can be used for rapidly detecting interaction among protein.
Description
Technical field
The present invention relates to reorganization fluorescence double cross yeast and detection method and application in the biological technical field.
Background technology
Yeast-two hybrid technique (yeast two hybrid) is the interactional experimental system of utilizing between yeast genetics methods analyst protein, is usually used in verifying interaction or the candidate albumen of screening and target protein specific action between known protein matter.Yeast-two hybrid technique has been widely used in a plurality of research fields such as cell cycle regulating, signal transduction, oncogene expression, pharmacokinetics.
Fields and Song have at first set up yeast two-hybrid system in research eukaryotic gene transcriptional control; This system is based on the complete understanding to yeast transcription factor GAL4, it have the territory of two function opposite independent: DNA combine the territory (DNA binding domain, BD) and transcription activating domain (activation domain; AD); BD can discern the upstream activating sequence of GAL4 effector (GAL4-responsive gene), and (upstream activatingsequence UAS), and combines with it.AD then through and transcriptional machinery (transcription machinery) in other compositions between effect, the gene that starts the UAS downstream is transcribed.The independent effect of BD and AD all can not the activated transcription reaction.And after both are spatially approaching, need form covalent linkage and get final product activated transcription.This just constitutes the basis of double cross system.
In the yeast two-hybrid system of classics, two kinds of proteinic genes to be studied are cloned into the downstream of the GAL4-AD and the GAL4-BD of Yeast expression carrier respectively, carry out amalgamation and expression, analyzing proteins is done mutually.Specifically; X and Y are two interactional albumen of the unknown; The fusion rotein of DB-X is called bait (bait); X is known protein normally, and AD-Y is called prey (prey), if bait and prey can express in host cell nuclear simultaneously; Then BD and AD form the transcriptional activation mixture, thereby activate the expression of
reporter gene.This expression of gene can detect through biochemical or genetic method and obtain, and can realize the research for protein (X and Y) interphase interaction.As researching and analysing interactional system between the protein-protein, the advantage of the maximum of yeast two-hybrid do not need to be separation and purification protein.Simultaneously, because yeast growth speed is fast, easy handling, this system has had simple and effective characteristics concurrently.
The reporter gene that yeast two-hybrid is commonly used at present has LacZ and nutrient defect type mark such as His etc., and the former can be through the color reaction screening positive clone of ONPG (or other substrates) when existing, and whether the latter then judges according to the growth on the screening culture medium.But as a kind of detection method, the auxotrophy mark can't carry out relative quantification to interactions between protein as reporter gene.And the detection of LacZ needs smudge cells usually, and temperature is bathed steps such as colour developing, complex operation, takes time and effort.These shortcomings have become the obstacle of double cross yeast high-throughput, rapid detection interactions between protein.
Summary of the invention
An object of the present invention is to provide a kind of fusion gene.
Fusion gene provided by the present invention, the fusion gene that connects and composes successively by the dna fragmentation shown in the 4336th to 5226 Nucleotide of sequence 5 in dna fragmentation, reporter gene, terminator, selection markers protein expression box and the sequence table shown in the 9th to 1030 Nucleotide of sequence 5 in the sequence table.
The nucleotides sequence of said terminator is classified as in the sequence table shown in the 2803rd to 3130 of the sequence 5;
Said reporter gene is a luciferase reporter gene;
Said selection markers protein expression box is a HIS Box expression cassette.
The nucleotide sequence of said luciferase reporter gene is shown in the 1063rd to 2715 of sequence 5 in the sequence table;
The nucleotides sequence of said HIS Box expression cassette is classified as in the sequence table shown in the 3152nd to 4329 of the sequence 5.
Said fusion gene is the dna molecular shown in the 9th to 5226 of the sequence 5 in the sequence table.
The recombinant plasmid that contains said fusion gene also belongs to protection scope of the present invention.
Another object of the present invention provides a kind of recombination microzyme.
Recombination microzyme provided by the present invention imports host yeast with said fusion gene and obtains; Said host yeast is the yeast of disappearance GAL4 gene and GAL80 gene.
Said host yeast is yeast Y187.
Another purpose of the present invention provides the method that detects uciferase activity in the said recombination microzyme.
The method of uciferase activity in the said recombination microzyme of detection provided by the present invention may further comprise the steps:
(1) makes luciferase expression in claim 6 or the 7 described recombination microzymes, obtain the recombination microzyme of luciferase expression;
(2) the said recombination microzyme that step (1) is obtained shakes cultivation, to the OD of bacterium liquid
600Value is 0.4-1.5 or 0.4 or 1.0 or 1.5, and the bacterium liquid that obtains is bacterium liquid to be measured; All material notes in the culture vessel are made bacterium liquid;
(3) the luciferase substrate is dissolved in obtains the luciferase substrate solution in the citric acid-sodium citrate damping fluid; The luciferase substrate solution that obtains is mixed with said bacterium liquid to be measured; Obtain mixture, detect the fluorescent value of mixture, promptly obtain said uciferase activity.
It is that to be 30 ℃ in temperature be to carry out under the condition of 200rpm with rotating speed that said concussion is cultivated, and rotation radius is 1.3cm; The substratum that said concussion is cultivated is an auxotrophy SD substratum.
The volume ratio of said bacterium liquid to be measured and said luciferase substrate solution is 1: 2-2: 1 or 1: 2 or 1: 1 or 2: 1;
The concentration of said luciferase substrate solution is 0.01mM;
Said luciferase substrate is the D-resorcinolphthalein;
The concentration of said citric acid-sodium citrate damping fluid is that 0.1mol/L and pH value are 3.0.
The application of said recombination microzyme in detecting protein-protein interaction also belongs to protection scope of the present invention.
Reorganization fluorescence yeast provided by the present invention can be used for the interaction between rapid detection protein.
Description of drawings
Fig. 1 is the fluoroscopic examination result of bacterium liquid to be measured under the different D-luciferin concentration.
Fig. 2 is the saccharomycetic growth curve of reorganization.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
One, makes up reorganization fluorescence yeast
1, gene fusion construct
(1) acquisition of each dna fragmentation in the fusion gene
A, the segmental acquisition of the full promoter region of GAL1
With yeast strain Y187 (available from Clontech company) genomic dna is template, and the design special primer carries out PCR, and acquisition obtains to contain the full promoter region of GAL1UAS and GAL1TATA sequence at the LacZ upper reaches, and concrete grammar is following:
The design primer sequence is following:
GAL-UP-NotI：
5′-A
GCGGCCGCCGACAGTTCCCAAAATG-3′，
GAL-UP-Hind：
5′-T
AAGCTTTATAGTTTTTTCTCCTTGACG-3′。
With yeast strain Y187 genomic dna is template, and the PCR reaction conditions is following:
94 ℃ 30 seconds
56 ℃ 30 seconds
72 ℃ 1 minute
30 circulations, last 72 ℃ were extended 5 minutes.
Through sequence verification, the sequence dna fragment that pcr amplification reaction obtains is a sequence 1 in the sequence table.The 591st of sequence 1 the-the 708th is the GAL1UAS sequence in the sequence table; The 1024th of sequence 1 the-the 1030th is the GAL1TATA sequence in the sequence table.Fragment called after GAL1UAS-GAL1TATA with the 1st the-the 1030th of sequence 1 in the sequence table.
B, LacZ-C hold pulsating acquisition
The design primer sequence is following:
LacZ-F2283B：
5′-A
GGATCCCTGACCACCAGCGAAATG-3′，
LacZ-R3167S：
5′-A
GTCGACGCGAAATACGGGCAGACAT-3′，
With yeast strain Y187 genomic dna is template, and the PCR reaction conditions is following:
94 ℃ 30 seconds
56 ℃ 30 seconds
72 ℃ 1 minute
30 circulations, last 72 ℃ were extended 5 minutes.
Through sequence verification, the sequence dna fragment that pcr amplification reaction obtains is a sequence 2 in the sequence table, and the 7th of sequence 2 the-the 897th is the part fragment of holding near C in the LacZ CDS sequence in the sequence table, with the dna fragmentation called after LacZ-C of this LacZ CDS sequence.
The acquisition of C, HIS Box expression casette
The design primer sequence is following:
HisBox-EcoR-UP：
5′-A
GAATTCATTCCCGTTTTAAGAGCTTG-3′，
HisBox-BamHI-DW：
5′-A
GGATCCTCGAGTTCAAGAGAAAAAAAAAGAAAAAGC-3′。
With plasmid pEG202 (available from Molecular Biotechnology) is template, and the PCR reaction conditions is following:
94 ℃ 30 seconds
56 ℃ 30 seconds
72 ℃ 1 minute
30 circulations, last 72 ℃ were extended 5 minutes.
Through sequence verification, the sequence dna fragment that pcr amplification reaction obtains is that the 7th the-the 1184th of sequence 3 is HIS Box sequence in the sequence table.
The acquisition of D, ADH terminator
The design primer sequence is following:
TADH-BamH-UP：
5′-A
GGATCCTGACTAGAATTAATGCCCA-3′，
TADH-EcoR-DW：
5′-A
GAATTCATTAGGTCCCTCGCACAT-3′。
With yeast strain Y187 genomic dna is template, and the PCR reaction conditions is following:
94 ℃ 30 seconds
56 ℃ 30 seconds
72 ℃ 1 minute
30 circulations, last 72 ℃ were extended 5 minutes.
Through sequence verification, the sequence dna fragment that pcr amplification reaction obtains is a sequence 4 in the sequence table, and the 81st of sequence 4 the-the 408th is ADH1 terminator sequence in the sequence table, with this ADH1 terminator sequence DNA fragment called after TADH.
(2) GAL1UAS-GAL1TATA links to each other with the Luc coding region
Luc coding region fragment is directly cut acquisition from pGL3-Basic plasmid (available from Promega company) with the HindIII/XbaI enzyme.
Reaction system is following:
pGL3-Basic 2ul(1ug/ul)
10 * multi-functional Buffer 2ul
HindIII 1ul(10u/ul)
XbaI 1ul(10u/ul)
ddH
2O 14ul
37℃ 4h
The above-mentioned about 1.6kb segment that obtains of electrophoretic separation is inserted into the HindIII/SpeI site of pBlue-SK carrier (available from general Jino Science and Technology Ltd., catalog number is pYB808),
Linked system is following:
Dna segment 2ul (1ug)
Carrier DNA 1ul (0.2ug)
10×ligation?Buffer?1ul
Ligase 1ul
ddH
2O 5ul
16 ℃ of connections are spent the night.
Get 5ul ligation liquid, add competence bacterium JM109 (available from precious biological, catalog number is D9052) 100ul, place 20min on ice, 42 ℃ of heat-shocked 2min, coating LB+Amp is dull and stereotyped after adding LB 1ml and cultivating 30min.
Provoke mono-clonal and identify with primer specificity PCR, obvious band appears in positive colony at the 1.6kb place.With the recombinant vectors called after pBlue-SK-Luc that obtains.
The dna fragmentation GAL1UAS-GAL1TATA that above-mentioned steps (1) is obtained is inserted into the NotI/HindIII site of recombinant vectors pBlue-SK-Luc,
Linked system is following:
Dna segment 2ul (1ug)
Carrier DNA 1ul (0.2ug)
10×ligation?Buffer 1ul
Ligase 1ul
ddH
2O 5ul
16 ℃ of connections are spent the night.
Get 5ul ligation liquid, add competence bacterium JM109100ul, place 20min on ice, 42 ℃ of heat-shocked 2min, coating LB+Amp is dull and stereotyped after adding LB 1ml and cultivating 30min.
Provoke mono-clonal and identify with primer specificity PCR, obvious band appears in positive colony at the 1.3kb place.With the recombinant vectors called after pBlue-SK-GAL1UAS-GAL1TATA-Luc that obtains.
(3) HIS Box expression cassette is linked to each other with LacZ-C terminal sequence fragment
Use the SmaI enzyme to cut respectively HIS Box expression cassette and LacZ-C terminal sequence fragment, obtain to have the fragment of identical sticky end.
Reaction system is following:
Dna fragmentation 2ul (1ug/ul)
10 * multi-functional Buffer 2ul
SmaI 1ul(10u/ul)
ddH
2O 15ul
37℃ 4h
The above-mentioned fragment that obtains is connected.
Linked system is following:
Dna fragmentation 2ul (1ug)
Carrier DNA 1ul (0.2ug)
10×ligation?Buffer 1ul
Ligase 1ul
ddH
2O 5ul
16 ℃ of connections are spent the night
Obtain the HIS-LacZ-C fragment, the EcoRI/SalI site that this fragment is inserted into recombinant vectors pBlue-SK-GAL1UAS-GAL1TATA-Luc forms following structure:
GAL1UAS-GAL1TATA-Luc-MCS-HIS-LacZ-C。
Linked system is following:
Dna fragmentation 2ul (1ug)
Carrier DNA 1ul (0.2ug)
10×ligation?Buffer 1ul
Ligase 1ul
ddH
2O 5ul
16 ℃ of connections are spent the night
Get 5ul ligation liquid, add competence bacterium JM109100ul, place 20min on ice, 42 ℃ of heat-shocked 2min, coating LB+Amp is dull and stereotyped after adding LB 1ml and cultivating 30min.
Provoke mono-clonal and identify with primer specificity PCR, obvious band appears in positive colony at the 2.1kb place.
(4) acquisition of fusion gene
The TADH fragment (BamHI/EcoRI) that step (1) is obtained is inserted between the Luc and HIS of the carrier that above-mentioned steps (3) obtains, the element order that final plasmid contains as follows: NotI-GAL1-Luc-ADHterminator-HIS-LacZ-C.Segmentation order-checking splicing result is sequence 5 in the sequence table.The the 9th to 1030 of sequence 5 is the full promoter region fragment of GAL1 in the sequence table, and wherein, the 591st the-the 708th of sequence 5 is the GAL1UAS sequence in the sequence table; The 1024th the-the 1030th of sequence 5 is the GAL1TATA sequence in the sequence table; The 1063rd the-the 2715th of sequence 5 is Luc CDS sequence in the sequence table; The 2803rd the-the 3130th of sequence 5 is the TADH sequence in the sequence table; The 3152nd the-the 4329th of sequence 5 is HIS Box sequence in the sequence table; The 4336th the-the 5226th of sequence 5 is the LacZ-C sequence in the sequence table.
2, make up recombination yeast
(1) the above-mentioned final plasmid of separation and purification with this plasmid of NotI/SalI double digestion, obtains the GAL1-Luc-ADHterminator-HIS-LacZ-C fragment, uses yeast conversion test kit (general Jino YGM011) transformed yeast bacterium Y187.Use empty carrier pBlue-SK transformed yeast bacterium Y187 simultaneously, obtain changeing empty carrier contrast yeast.
(2) the picking mono-clonal is cultivated in-His substratum, and the picking positive colony extracts the genomic dna of positive colony.Primer of resynthesis carries out the positive colony evaluation at the segmental upper reaches far away of GAL1 upstream activating sequence.Primer sequence is following:
GAL1-far-up：5’-CTCGTAGCATCACCATAGA-3’，
Carrying out PCR with above-mentioned primer and the pairing of Luc629R sequence identifies:
Luc629R：5’-AGAGCGACACCTTTAGGCAGA-3’。
The PCR qualification result shows: the PCR product of 1.7kb band appears in positive colony, changes the empty carrier contrast and does not have this band generation.
(3) extract positive colony genomic dna segmentation amplification regional area sequencing fragment and confirm that sequencing result is consistent with implementation sequence.The final yeast genes type that makes up is MAT α, ura 3-5, ade 2-101 trp 1-901, leu 2-3,112, gal4 Δ, met-, gal80 Δ, URA::GAL1UAS-GAL1TATA-Luc.This recombination yeast is a skeleton with yeast strain Y187, and the Luc gene is integrated into that the GAL1UAS-GAL1TATA downstream are regulated and control by it in the yeast chromosomal.Identical with Y187, this zymic GAL4 gene and GAL80 gene (Gal80 is the negative regulatory factor of Gal4) are lacked, thereby have got rid of the influence of the endogenous regulatory factor of cell.Different with Y187, this yeast has the HIS synthesis capability.This yeast is based on the GAL4 system, and all known these system's plasmids all can duplicate in this host or express as required at present.
Two, fluorescence double cross yeast functional verification
1, draws growth curve
(1) conversion of positive control plasmid
But use in yeast conversion test kit or the LiAc normal direction reorganization fluorescence yeast and transform the proteic plasmid pCL1 of The expressed GAL4 (available from Promega company, catalog number is K1604-1).This albumen is wild-type GAL4 albumen, can activate GAL4 response element (UAS), and then starts the expression of this element downstream reporter gene.PCL1 is usually as the positive control plasmid of GAL4 two-hybrid system.GAL1UAS-GAL1TATA-Luc fragment biological activity in this recombinates fluorescence yeast genes group in order to detection.
(2) screening of positive colony
With the bacterium liquid after transforming coat auxotrophy solid SD dull and stereotyped (Leu ,-His), be inverted to cultivate for 30 ℃ and occurred to single spot in 1-2 days.
(3) fluoroscopic examination
Method I
Picking has the yeast list spot of Trp and His synthesis capability, with auxotrophy SD substratum (Leu ,-His) shake cultivation (30 ℃, 200rpm, rotation radius is 1.3em), to the OD of bacterium liquid
600Value is 0.4, and the bacterium liquid that obtains is bacterium liquid to be measured; All material notes in the culture vessel are made bacterium liquid.
Auxotrophy SD substratum (Leu ,-His) prescription is: 20mgL-1 VITAMIN B4 Hemisulphate, 20mgL-1 arginine hydrochloride, 20mgL-1 tryptophane; The 30mgL-1 Isoleucine, 30mgL-1 lysine hydrochloride, 20mgL-1 methionine(Met); The 50mgL-1 phenylalanine(Phe), 200mgL-1 Threonine, 30mgL-1 tyrosine; The 20mgL-1 uridylic, 150mgL-1 Xie Ansuan, 6.7gL-1 do not have the amino acid yeast nitrogen.
D-resorcinolphthalein (D-luciferin) is dissolved in the citric acid-sodium citrate damping fluid (the pH value is 3.0); Obtain D-luciferin solution (establish the D-luciferin solution of three concentration: 1mM, 0.1mM and 0.01mM); 200ul D-luciferin solution is mixed with 100ul bacterium liquid to be measured; Obtain mixture, will obtain mixture and add check-out console (in vain), detect the fluorescent value of mixture.The multi-functional ELIASA of TECAN Infinite M200 is used in fluorometric assay, and 90 every 2min of circulation measure once, and be 1s integral time, linear concussion 2s before each mensuration the, concussion diameter 4mm.
The compound method of citric acid-sodium citrate damping fluid is following:
0.1mol/L Hydrocerol A 18.6ml is mixed the back obtain 20ml citric acid-sodium citrate damping fluid with 0.1mol/L Trisodium Citrate 1.4ml, the pH value is 3.0, and concentration is 0.1mol/L.
The result: experimental result is as shown in Figure 1, and fluorescent signal was comparatively stable when the concentration of D-luciferin was 0.01mM, and it is less to reach variation of temperature in time.And, use the substrate of lower concentration to have bigger meaning for reducing experimental cost.So confirm to use the D-luciferin of 0.01mM to detect.
According to OD600 value and the luminous value measured, draw growth curve (Fig. 2).
The result shows that this recombination yeast has 7-8 hour logarithmic phase through behind the lag phase, through beginning decline after 5-4 hour stationary phase.It has been generally acknowledged that logarithmic growth is interim, yeast growth speed is the fastest, metabolism is vigorous, enzyme system is active, the chemical constitution form physico-chemical property basically identical of cell.Quantitative examination suits to carry out.
Method II
Except that following 1) and 2) method, all the other methods are all identical with method I.
(1) to the OD of bacterium liquid
600Value is 1.0.
(2) 100ul D-luciferin solution is mixed with 100ul bacterium liquid to be measured.
The result: I does not have significant difference with method.
Method III
Except that following 1) and 2) method, all the other methods are all identical with method I.
(1) to the OD of bacterium liquid
600Value is 1.5.
(2) 50ul D-luciferin solution is mixed with 100ul bacterium liquid to be measured.
The result: I does not have significant difference with method.
PGBKT7-53 plasmid (available from clontech company, catalog number is 630303) comprises fusion gene GAL4DNA-BD-murine p53; PGADT7-T plasmid (available from Clontech company, catalog number is 630303) comprises fusion gene GAL4AD-large T-antigen.Wherein albumen murine p53 and large T-antigen can interact.
One, plasmid transforms
Cotransformation comprises the pGBKT7-53 plasmid and the pGADT7-T plasmid that comprises fusion gene GAL4AD-largeT-antigen of fusion gene GAL4DNA-BD-murine p53 in the reorganization fluorescence yeast that use yeast conversion test kit or LiAc normal direction embodiment 1 make up.Because the two is expressed simultaneously, albumen murine p53 and large T-antigen can interact, and make BD and AD spatially approaching, form the transcriptional activation mixture, can activate the expression of GAL4 response element (UAS) and then activation reporter gene luciferase.Can realize research for protein (X and Y) interphase interaction.
PGBKT7-53 plasmid and pGADT7-T plasmid are transformed reorganization fluorescence yeast respectively separately as negative control, the pCL1 plasmid is transformed reorganization fluorescence yeast as positive control, the reorganization fluorescence yeast of establishing unconverted plasmid simultaneously is for changeing the empty carrier contrast.
It is dull and stereotyped that bacterium liquid after transforming is coated auxotrophy solid SD, is inverted 1-2 days extremely single spots of cultivation for 30 ℃ and occurs.Picking yeast list spot, incubated overnight (30 ℃, 200rpm) after, use the specific primer of pGBKT7-53 plasmid and pGADT7-T plasmid to carry out bacterium liquid PCR and verify.
The pGBKT7-53 primer sequence is following:
T7sequencing?primer：
5’-TAATACGACTCACTATAGGGCG-3’
3’DNA-BD?sequencing?primer
5’-TTTTGCTTTTAAAACCTAAGAGTC-3’
Positive findings bright band occurs at 0.6kb.
The pGADT7-T primer sequence is following
T7sequencing?primer：
5’-TAATACGACTCACTATAGGGCG-3’
3’AD?sequencing?primer：
5’-TCTACCACGTGCTACGTGTC-3’
Positive findings bright band occurs at 0.8kb.
Two, fluoroscopic examination
The positive yeast list of picking spot, with auxotrophy SD substratum (Leu ,-Trp) shake cultivation (30 ℃, 200rpm, rotation radius is 1.3cm), to the OD of bacterium liquid
600Value is 0.4, and the bacterium liquid that obtains is bacterium liquid to be measured, and all the material notes in the culture vessel are made bacterium liquid, can carry out fluorescence radiation and detect.
Auxotrophy SD substratum (Leu ,-Trp) prescription is: 20mgL-1 VITAMIN B4 Hemisulphate, 20mgL-1 arginine hydrochloride, 20mgL-1 one hydration histidine monohydrochloride; The 30mgL-1 Isoleucine, 30mgL-1 lysine hydrochloride, 20mgL-1 methionine(Met); The 50mgL-1 phenylalanine(Phe), 200mgL-1 Threonine, 30mgL-1 tyrosine; The 20mgL-1 uridylic, 150mgL-1 Xie Ansuan, 6.7gL-1 do not have the amino acid yeast nitrogen.
Fluorescence analysis is identical with the method I of step 2 among the embodiment 1.
The fluoroscopic examination result is as shown in table 1:
Table 1 transform different plasmids reorganization fluorescence yeast liquid fluorometric assay result
| Contain plasmid | Relative intensity of fluorescence |
| -(changeing the empty carrier contrast) | 26±18 |
| PCL1 (positive control) | 18951±61 |
| PGBKT7-53 (negative control) | 20±16 |
| PGADT7-T (negative control) | 31±15 |
| PGBKT7-53 and pGADT7-T | 15800±54 |
Annotate: relative intensity of fluorescence=fluorescent value/OD
600
Visible from table 1; The reorganization fluorescence yeast of cotransformation pGBKT7-53 plasmid and pGADT7-T plasmid all detects higher fluorescent signal with the positive control that transforms the pCL1 plasmid, all detects more weak fluorescent signal and transform the reorganization fluorescence yeast of pGBKT7-53 plasmid and pGADT7-T plasmid separately and change the empty carrier contrast.This presentation of results, the reorganization fluorescence yeast that utilizes the present invention to make up can be used for detecting the interaction between the protein.
Claims (6)
1. fusion gene is the dna molecular shown in the 9th to 5226 of the sequence 5 in the sequence table.
2. the recombinant plasmid that contains the said fusion gene of claim 1.
3. a recombination microzyme imports host yeast with the said fusion gene of claim 1 and obtains; Said host yeast is yeast Y187.
4. test right requires the method for uciferase activity in the 3 described recombination microzymes, may further comprise the steps:
(1) makes luciferase expression in the described recombination microzyme of claim 3, obtain the recombination microzyme of luciferase expression;
(2) the said recombination microzyme that step (1) is obtained shakes cultivation, to the OD of bacterium liquid
600Value is 0.4-1.5, and the bacterium liquid that obtains is bacterium liquid to be measured; All material notes in the culture vessel are made bacterium liquid;
(3) the luciferase substrate is dissolved in obtains the luciferase substrate solution in the citric acid-sodium citrate damping fluid; The luciferase substrate solution that obtains is mixed with said bacterium liquid to be measured; Obtain mixture, detect the fluorescent value of mixture, promptly obtain said uciferase activity.
5. method according to claim 4 is characterized in that:
It is that to be 30 ℃ in temperature be to carry out under the condition of 200rpm with rotating speed that said concussion is cultivated, and rotation radius is 1.3cm;
The volume ratio of said bacterium liquid to be measured and said luciferase substrate solution is 1: 2-2: 1;
The concentration of said luciferase substrate solution is 0.01mM;
Said luciferase substrate is the D-resorcinolphthalein;
The concentration of said citric acid-sodium citrate damping fluid is that 0.1mol/L and pH value are 3.0.
6. the application of the described recombination microzyme of claim 3 in detecting protein-protein interaction.
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101469315B (en) * | 2007-12-29 | 2011-03-30 | 中国科学院生态环境研究中心 | Two-hybrid yeast for detecting estrogen-like compound in environment and biological test method |
| CN101469314B (en) * | 2007-12-29 | 2011-04-06 | 中国科学院生态环境研究中心 | Two-hybrid yeast and method for detecting thyroid hormone-like/antithyroid hormone compound |
| CN101469316B (en) * | 2007-12-29 | 2011-06-29 | 中国科学院生态环境研究中心 | Two-hybrid yeast and biological test method for detecting retinoic acid-like and retinoic acid resistant compound |
Non-Patent Citations (4)
| Title |
|---|
| JIAN LI ET AL.A two-hybrid yeast assay to quantify the effects of xenbotics on thyroid hormone-mediated gene expression.《Enviromental Toxicology and Chemistry》.2008,159-167. * |
| 李剑等.核受体超家族及其酵母双杂交检测技术.《生态毒理学报》.2008,521-530. * |
| 段强德,陈铁桥.酵母双杂交体系的原理及研究进展.《畜牧兽医杂志》.2009,56－58. * |
| 秦少溥.酵母双杂交及其衍生系统.《生物学通报》.2009,14-18. * |
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| CN102127561A (en) | 2011-07-20 |
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