CN107043764A - A kind of method that the yellow chicken interaction of genes albumen in Rugao is found based on GST Pull Down and analytical technique of mass spectrum - Google Patents
A kind of method that the yellow chicken interaction of genes albumen in Rugao is found based on GST Pull Down and analytical technique of mass spectrum Download PDFInfo
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Abstract
The invention discloses a kind of method that the yellow chicken interaction of genes albumen in Rugao is found based on GST Pull Down and analytical technique of mass spectrum：（1）Target gene is cloned and Prokaryotic expression vector construction；（2）The prokaryotic fusion expression vector of Pet 49 (b) GST target gene is built；（3）Pet 49 (b) GST target gene carries out prokaryotic expression；（4）Great expression and the detection of broken bacterium are carried out to albumen；（5）Specify and the albumen of expression is purified and concentrated behind the expression position of albumen and is carried out GST Pull Down experiments；（6）Albumen after GST Pull Down carries out protein reductive alkylation and film dosim and the sequence for the interaction albumen for obtaining target gene is identified by LC MS/MS.The present invention can obtain the interaction albumen of related gene faster；Method is simple and easy to apply, and repeatability is strong, can be carried out in common lab.
Description
Technical field
The present invention relates to the association areas such as molecular biology engineering.
Technical background
GST Pull-Down experiments are the in vitro test technologies of an effective checking yeast two-hybrid system, closely
Increasingly favored over year by numerous scholars.Its general principle is to be solidificated in gluathione by target protein-gst fusion protein is affine
In peptide affine resin, as the supporter affine with destination protein, a kind of bait protein is served as, destination protein solution is crossed into post,
The interaction therefrom captured between the destination protein interacted therewith, albumen can be by SDS-PAGE electrophoresis and other phases
The detection method of pass confirms that these methods include Coomassie brilliant blue, silver or zinc dyeing, Western-blotting and radiation
Property isotope 32S detection etc..Li Rong et al. confirms the phase interaction with KyTo and RBP-J by exchanging carrier in yeast first
With.And then KyoT2 albumen has been purified in expression in escherichia coli, and the polyclonal Hangzhoupro bodies of KyoT2 are prepared for as antigen, pass through
GST-pull down test further the interaction demonstrated in Escherichia coli in vitro between KyoT2 and interaction albumen.Lu Ling
Cloud et al. demonstrates ETAR C-terminals and FLJ using GST pull-down and interacts in vitro and inquired into ETAR and ETBR
With the interaction of unknown protein molecule so that illustrate its to downstream signal adjust effect.
The content of the invention
The yellow chicken base in Rugao is found based on GST-Pull Down and analytical technique of mass spectrum it is an object of the invention to provide one kind
Because of the method for interaction albumen, the mutual of the yellow chicken target gene in molecular biology method fast searching Rugao can be passed through using this method
Make albumen, this method is not applied still at present on chicken at present.
The purpose of the present invention is achieved through the following technical solutions, and one kind is based on GST-Pull Down and mass spectrum point
The method that analysis technology finds the yellow chicken interaction of genes albumen in Rugao, comprises the following steps：
(1) target gene clone and Prokaryotic expression vector construction
The CDS complete sequences of target gene are found according to the NCBI genes number of logging in of the yellow chicken target gene in Rugao, design is special
Property primer simultaneously introduces EcoR I and Hind III digestions site, and restriction enzyme site is determined according to the essential information of target gene,
Target gene CDS complete sequences are cloned by PCR method, through sequence verification after clone products purifying, for follow-up test；
(2) prokaryotic fusion expression vector of Pet-49 (b)-GST- target gene is built
By step (1) be sequenced correct clone products and Pet-49 (b) prokaryotic expression carriers respectively by EcoR I and
Hind III purified and connected after double digestion, digestion 3h, and DH5 α, picking positive bacteria clone are converted after connection 16h
And plasmid is extracted, acquisition, which is successfully constructed, to be identified to the pronucleus recombinant expression of structure by double digestion and sequencing
Pet-49 (b)-GST- target gene plasmids；
(3) Pet-49 (b)-GST- target gene carries out prokaryotic expression
Step (2) is obtained to Pet-49 (the b)-GST- target gene plasmid conversion protokaryon successfully constructed and reaches strain BL, together
Shi Liyong isopropylthiogalactosides IPTG is induced BL strains, is extracted bacterium solution albumen after induction 3h, is passed through SDS-
PAGE detects the expression of albumen, while utilizing GST and the expression of target gene gene antibody testing goal albumen；
(4) great expression is carried out to albumen and broken bacterium detects；
(5) albumen of expression is purified and concentrated behind the expression position of clear and definite albumen and carry out GST-Pull Down
Experiment；
(6) albumen after GST-Pull Down carries out protein reductive alkylation and film dosim and passes through LC-MS/MS
Identification obtains the sequence of the interaction albumen of target gene.
It is preferred that, step (4) the albumen great expression and broken bacterium detection detailed process are：
By taking 300ml as an example, 3mLLB fluid nutrient mediums are taken, antibiotic is added；Bacterium solution albumen thaw at RT is taken, is fully mixed
Afterwards, 3 μ L are taken to be inoculated with the LB culture mediums；37 DEG C of shaking table is placed in, 200rpm shakes bacterium overnight；By the bacterium solution of 3mL overnight growths
LB culture mediums of the 300mL containing antibiotic is all inoculated in, 37 DEG C, 200rpm growths are cooled to after required inducing temperature and added
Enter isopropylthiogalactoside IPTG to be induced；Bacterium solution is poured into 50mL centrifuge tubes after 4 hours, 4 DEG C of 8000rpm centrifugations
3min, abandons supernatant；Thalline adds 25mL buffer solutions to be resuspended, and loads in 25mL beakers；Plus lysozyme is mixed, VLysozyme：VBacterium solution=1:200；
30min on ice；It is ultrasonic on ice, the working time：3h；Off time：4s；Work times：99；Power is no more than 200w；Pour into from
In heart pipe, 4 DEG C of 16000rpm centrifuge 30min；Sample is taken to carry out SDS-PAGE detections；Remaining supernatant and precipitation are standby as 0-7 DEG C
With.
It is preferred that, the step (5) is purified and concentrated supernatant protein purifying detailed process to the albumen of expression and is：
GST Ago-Gels are loaded into chromatographic column, rinsed with the Buffer of 10 times of bed volumes；Step (4) is detected
Supernatant samples are added in chromatographic column, and flow control is in 0.5mL/min；Chromatograph and rinse removal of impurities with the Buffer of 10 times of bed volumes,
Flow control is in 1mL/min；With the GSH buffer elutions of 10 times of bed volumes, flow control collects eluting peak in 1mL/min；
SDS-PAGE runs glue detection each component；Carry out protein concentration.
It is preferred that, the step (5) carries out GST pull down experiments detailed process and is：
Draw μ L, the 3000rpm centrifugations 5min of glutathione-Sepharose beads suspension 500 and abandon supernatant, phosphate buffer PBS
Cleaning precipitation 3 times, is finally resuspended in 500 μ L PBS by precipitation, obtains suspension；1mL gst fusion proteins lysate is added into institute
State in suspension, 1h is incubated in 4 DEG C of Mute mixers, 3000rpm centrifugations abandon supernatant, then add another 500ul GST and melt
1h is incubated in hop protein lysate, 4 DEG C of Mute mixers, 3000rpm centrifugations 5min abandons supernatant, repeats this step 3-4 times, make
Enough gst fusion proteins are combined on sepharose pearls；With the 1mL PBS sepharose pearls 2 times of precooling, it is resuspended in
In 500 μ L PBS suspensions；Added in sepharose pearls Jing Yin mixed in 30 μ L testing sample protein lysates RAPI, 4 DEG C of chromatography cabinets
5h is incubated in clutch；3500rpm centrifuges 5min, carefully absorbs supernatant；The PBS that 500 μ L contain 1%Triton is often added in pipe clear
Precipitation is washed, is repeated 2 times, finally precipitation is resuspended in 200 μ L PBS；Draw 20 μ L and obtain precipitation, add 4 μ L 6xloading
Buffer boils 5min, and centrifugation draws supernatant and carries out SDS-PAGE electrophoresis.
It is preferred that, step (6) the protein reductive alkylation and film dosim detailed process are：
The protein after 100 μ gSDS-PAGE electrophoresis is taken out, final concentration 10mM dithiothreitol (DTT)s reduction egg is then added
White matter, is subsequently added into final concentration 55mM iodoacetic acid ammoniums, is eventually adding 1 μ g trypsase, and 8h~16h is digested overnight；Enzymolysis production
Raw polypeptide C18 pillar desalinations, use loading buffer solution polypeptide after the polypeptide of desalination is drained, sample-loading buffer is effective
Composition：0.1% formic acid, 3% acetonitrile, the final concentration of the dissolving of polypeptide is 1 μ g/ μ L.
It is preferred that, step (6) described LC-MS/MS identifies that detailed process is：
LC-MS/MS (ekspertTM nanoLC on polypeptide；AB Sciex Triple TOF 5600-plus) instrument enters
Row analysis, analytical column is AB SCIEX analytical columns, and the specification of AB SCIEX analytical columns is 75 μm of internal diameters, fills 3 μm,'s
ChromXP C18 post material, long 10cm, nozzle needle is NEW objective, and the specification of nozzle needle is 20 μm of internal diameters, the diameter of nozzle needle mouthful
It is 10 μm, trapping column is eksigent Chromxp Trap Column, 3 μm of C18-CL of specification of trapping column, 350μm
×0.5mm。
It is preferred that, the spectrogram qualification result of the protein digestion polypeptide is shown in Table 1
Machine data analysis parameter under the LC-MS/MS of the polypeptide of table 1
It is preferred that, design different inducing temperatures and isopropylthiogalactoside in step (3) described Induction Process
IPTG induced concentration.
It is preferred that, Pet-49 (b) prokaryotic expression carrier is purchased from BioVector plasmid vector bacterium cell gene preservations
Center.
It is preferred that, specific primer passes through Primer5.0 Software for Design in the step (2).
Compared with prior art, it is of the invention to be advantageous in that：
1st, the interaction albumen of related gene can be obtained faster.
2nd, method is simple and easy to apply, and repeatability is strong, can be carried out in common lab.
3rd, this method experiment in vitro is applied to other species.
Embodiment
A kind of method that the yellow chicken interaction of genes albumen in Rugao is found based on GST-Pull Down and analytical technique of mass spectrum,
A kind of method that the yellow chicken interaction of genes albumen in Rugao is found based on GST-Pull Down and analytical technique of mass spectrum：According to purpose
The NCBI genes number of logging in of gene finds the CDS complete sequences of target gene, by Primer5.0 Software for Design specific primer simultaneously
EcoR I and Hind III (restriction enzyme site can be determined according to the essential information of gene) restriction enzyme site is introduced, passes through PCR
Method clones target gene CDS total lengths, through sequence verification after clone products purifying, for follow-up test；Correct gram will be sequenced
Grand product and Pet-49 (b) prokaryotic expression carrier carries out carrying out after double digestion, digestion 3h respectively by EcoR I and Hind III
Purify and connect, convert DH5 α after connection 16h, picking positive bacteria is cloned and extracts plasmid, by double digestion and sequencing to structure
The pronucleus recombinant expression built is detected；Obtain Pet-49 (the b)-GST- target gene recombinant plasmids successfully constructed
And BL expression strains are converted, while being induced using IPTG BL strains.Design in the process different inducing temperatures and
Bacterium solution albumen is extracted after IPTG induced concentration, induction 3h, the expression of albumen is detected by SDS-PAGE, is utilized simultaneously
GST and target gene gene antibody testing goal albumen expression；It is right after inducing temperature and IPTG induced concentration to specify
Albumen carries out great expression and the detection of broken bacterium；Specify and the albumen of expression is purified behind the expression position of albumen and concentration is gone forward side by side
Row GST-Pull Down are tested；Albumen after GST-Pull Down carries out protein reductive alkylation and film dosim and passed through
LC-MS/MS identifications obtain the sequence of the interaction albumen of target gene.Specifically include following steps：
(1) target gene clone and Prokaryotic expression vector construction
The CDS complete sequences of target gene are found according to the NCBI genes number of logging in of target gene, pass through Primer5.0 softwares
Design specific primer simultaneously introduces EcoR I and Hind III (restriction enzyme site can be determined according to the essential information of gene)
Restriction enzyme site, target gene CDS total lengths are cloned by PCR method, through sequence verification after clone products purifying, for subsequently trying
Test；
(2) prokaryotic fusion expression vector of Pet-49 (b)-GST- target gene is built
Correct clone products and Pet-49 (b) prokaryotic expression carriers will be sequenced respectively by EcoR I and Hind III to enter
Purified and connected after row double digestion, digestion 3h, convert DH5 α after connection 16h, picking positive bacteria is cloned and extracts plasmid,
The pronucleus recombinant expression of structure is detected by double digestion and sequencing；
(3) Pet-49 (b)-GST- target gene carries out prokaryotic expression
Obtain Pet-49 (the b)-GST- target gene recombinant plasmid successfully constructed and convert into BL expression strains, simultaneously
BL strains are induced using IPTG；Different inducing temperature and IPTG induced concentration are designed in the process, induce 3h
Bacterium solution albumen is extracted afterwards, the expression of albumen is detected by SDS-PAGE, while being examined using GST and target gene gene antibody
Survey the expression of destination protein；
(4) albumen great expression and the detection of broken bacterium are (exemplified by 300ml)
The LB fluid nutrient mediums of a pipe 3mL are taken, corresponding antibiotic is added；Bacterium solution thaw at RT is taken, after fully mixing, is taken
3 μ L are seeded to above-mentioned culture medium；37 DEG C of shaking table is placed in, 200rpm shakes bacterium overnight；The bacterium solution of 3mL overnight growths is all inoculated with
Added in 300mL in the LB culture mediums of corresponding antibiotic, 37 DEG C, 200rpm growths are cooled to after required inducing temperature
Optimum concentration IPTG is added to be induced；Bacterium solution is poured into 50mL centrifuge tubes after 4 hours, 8000rpm centrifugation (4 DEG C) 3min,
Abandon supernatant；Thalline adds 25mL accordingly to buffer resuspension, loads in 25mL beakers；Plus lysozyme (1:200) mix, on ice 30min；Ice
Upper ultrasound (the working time：3h；Off time：4；Work times：99；Power is no more than 200w)；Pour into centrifuge tube,
16000rpm centrifuges (4 DEG C) 30min；A small amount of sample is taken to carry out SDS-PAGE detections；Remaining supernatant and precipitation are standby as 0-7 DEG C
With；
(5) supernatant protein is purified
GST Ago-Gels are loaded into suitable chromatographic column, rinsed with the Buffer of 10 times of bed volumes；It will detect above
Supernatant samples be added in chromatographic column, flow control is in 0.5mL/min or so；Chromatograph and rinsed with the Buffer of 10 times of bed volumes
Removal of impurities, flow control is in 1mL/min or so；With the GSH buffer elutions of 10 times of bed volumes, flow control is left in 1mL/min
The right side, collects eluting peak；SDS-PAGE runs glue detection each component；Carry out protein concentration；
(6)GST pull down
Draw μ L, the 3000rpm centrifugations 5min of glutathione-sepharose pearls suspension 500 and abandon supernatant, PBS sinks
Form sediment 3 times, precipitation is finally resuspended in 500 μ L PBS；1mL gst fusion proteins lysate is added into suspension obtained in the previous step
In, 1h is incubated in 4 DEG C of Mute mixers, 3000rpm centrifugations abandon supernatant, then add another gst fusion protein lysate,
1h is incubated in 4 DEG C of Mute mixers, 3000rpm centrifugations 5min abandons supernatant, repeats this step 3-4 times, makes to tie on sepharose pearls
Close enough gst fusion proteins；By the 1mL PBSs 2 times of sepharose pearls precooling obtained in the previous step, 500 are resuspended in
In μ L PBS suspensions；30 μ L testing sample protein lysates, 4 DEG C of chromatography cabinets are added in the sepharose pearls obtained one step up
5h is incubated in middle Mute mixer；350 0rpm centrifuge 5min, carefully absorb supernatant；500 μ L are often added in pipe and contain 1%
Triton PBS precipitation, is repeated 2 times, finally precipitation is resuspended in 200 μ L PBS；Draw 20 μ L and obtain precipitation, add
4 μ L 6xloading buffer boil 5min, and centrifugation draws supernatant and carries out SDS-PAGE electrophoresis；(7) protein reductive alkylation
And film dosim
The reductive alkylation of protein is as follows：100 μ g proteins are taken out, then final concentration 10mM dithiothreitol (DTT)s are added also
Crude protein, is subsequently added into final concentration 55mM iodoacetic acid ammonium (IAM), is eventually adding 1 μ g Trypsin enzymes, overnight digest 8h~
16h；The polypeptide C18 pillar desalinations produced are digested, Loading Buffer (0.1% first is used after the polypeptide of desalination is drained
Acid, 3% acetonitrile) dissolving polypeptide, the final concentration of the dissolving of polypeptide is 1 μ g/ μ L；
(8) the LC-MS/MS identifications of protein digestion polypeptide
LC-MS/MS (ekspertTM nanoLC on polypeptide；AB Sciex Triple TOF 5600-plus) instrument enters
Row analysis, analytical column be AB SCIEX analytical columns (75 μm of internal diameters, fill 3 μm,ChromXP C18 post material, it is long
10cm), nozzle needle is NEW objective (20 μm of internal diameters, the diameter of nozzle needle mouthful is 10 μm), and trapping column is eksigent
Chromxp Trap Column(3μm C18-CL,350μm×0.5mm)；
(9) the spectrogram identification of protein digestion polypeptide
Machine data analysis parameter under the LC-MS/MS of polypeptide
The invention of this method, is found such as mainly for providing one kind based on GST-Pull Down and analytical technique of mass spectrum
The method of highland Huang chicken interaction of genes albumen.
Using this method, can by the interaction albumen of the yellow chicken target gene in molecular biology method fast searching Rugao,
This method is not applied still at present on chicken at present.
The present invention is advantageous in that：
1st, the interaction albumen of related gene can be obtained faster.
2nd, method is simple and easy to apply, and repeatability is strong, can be carried out in common lab.
3rd, this method experiment in vitro is applied to other species.
Claims (10)
1. a kind of method that the yellow chicken interaction of genes albumen in Rugao is found based on GST-Pull Down and analytical technique of mass spectrum, its
It is characterized in that the described method comprises the following steps：
(1) target gene clone and Prokaryotic expression vector construction
The CDS complete sequences of target gene are found according to the NCBI genes number of logging in of the yellow chicken target gene in Rugao, design specificity is drawn
Thing simultaneously introduces EcoR I and Hind III digestions site, and restriction enzyme site is determined according to the essential information of target gene, passed through
PCR method clones target gene CDS complete sequences, through sequence verification after clone products purifying, for follow-up test；
(2) prokaryotic fusion expression vector of Pet-49 (b)-GST- target gene is built
Correct clone products are sequenced in step (1) and Pet-49 (b) prokaryotic expression carriers pass through EcoR I and Hind respectively
III purified and connected after double digestion, digestion 3h, converts DH5 α after connection 16h, picking positive bacteria is cloned and extracted
Plasmid, is identified the pronucleus recombinant expression of structure by double digestion and sequencing, obtains the Pet- successfully constructed
49 (b)-GST- target gene plasmids；
(3) Pet-49 (b)-GST- target gene carries out prokaryotic expression
Step (2) is obtained to Pet-49 (the b)-GST- target gene plasmid conversion protokaryon successfully constructed and reaches strain BL, while sharp
BL strains are induced with isopropylthiogalactoside IPTG, bacterium solution albumen is extracted after induction 3h, is examined by SDS-PAGE
The expression of albumen is surveyed, while utilizing GST and the expression of target gene gene antibody testing goal albumen；
(4) great expression is carried out to albumen and broken bacterium detects；
(5) albumen of expression is purified and concentrated behind the expression position of clear and definite albumen and carry out GST-Pull Down experiments；
(6) albumen after GST-Pull Down carries out protein reductive alkylation and film dosim and identified by LC-MS/MS
Obtain the sequence of the interaction albumen of target gene.
2. it is according to claim 1 a kind of mutual based on the yellow chicken gene of GST-Pull Down and mass spectral analysis searching Rugao
Make the method for albumen, it is characterized in that, step (4) the albumen great expression and broken bacterium detection detailed process are：
By taking 300ml as an example, 3mLLB fluid nutrient mediums are taken, antibiotic is added；Bacterium solution albumen thaw at RT is taken, after fully mixing, is taken
3 μ L are inoculated with the LB culture mediums；37 DEG C of shaking table is placed in, 200rpm shakes bacterium overnight；The bacterium solution of 3mL overnight growths is whole
Be inoculated in LB culture mediums of the 300mL containing antibiotic, 37 DEG C, 200rpm growths, cool to added after required inducing temperature it is different
Propyl dithiocarbamate galactoside IPTG is induced；Bacterium solution is poured into 50mL centrifuge tubes after 4 hours, 4 DEG C of 8000rpm centrifugations
3min, abandons supernatant；Thalline adds 25mL buffer solutions to be resuspended, and loads in 25mL beakers；Plus lysozyme is mixed, VLysozyme：VBacterium solution=1:200；
30min on ice；It is ultrasonic on ice, the working time：3h；Off time：4s；Work times：99；Power is no more than 200w；Pour into from
In heart pipe, 4 DEG C of 16000rpm centrifuge 30min；Sample is taken to carry out SDS-PAGE detections；Remaining supernatant and precipitation are standby as 0-7 DEG C
With.
3. it is according to claim 2 a kind of mutual based on the yellow chicken gene of GST-Pull Down and mass spectral analysis searching Rugao
Make the method for albumen, it is characterized in that, the step (5) is purified and concentrated supernatant protein to the albumen of expression and purifies specific mistake
Cheng Wei：
GST Ago-Gels are loaded into chromatographic column, rinsed with the Buffer of 10 times of bed volumes；The supernatant that step (4) is detected
Sample is added in chromatographic column, and flow control is in 0.5mL/min；Chromatograph and rinse removal of impurities, flow velocity with the Buffer of 10 times of bed volumes
Control is in 1mL/min；With the GSH buffer elutions of 10 times of bed volumes, flow control collects eluting peak in 1mL/min；SDS-
PAGE runs glue detection each component；Carry out protein concentration.
4. it is according to claim 1 a kind of mutual based on the yellow chicken gene of GST-Pull Down and mass spectral analysis searching Rugao
Make the method for albumen, it is characterized in that, the step (5) carries out GST pull down experiments detailed process and is：
Draw μ L, the 3000rpm centrifugations 5min of glutathione-Sepharose beads suspension 500 and abandon supernatant, phosphate buffer PBS
Precipitation 3 times, is finally resuspended in 500 μ L PBS by precipitation, obtains suspension；1mL gst fusion proteins lysate is added described outstanding
In liquid, 1h is incubated in 4 DEG C of Mute mixers, supernatant is abandoned in 3000rpm centrifugations, then adds another 500ul GST fusion eggs
1h is incubated in white lysate, 4 DEG C of Mute mixers, 3000rpm centrifugations 5min abandons supernatant, repeats this step 3-4 times, make
Enough gst fusion proteins are combined on sepharose pearls；With the 1mL PBS sepharose pearls 2 times of precooling, it is resuspended in
In 500 μ L PBS suspensions；Added in sepharose pearls Jing Yin mixed in 30 μ L testing sample protein lysates RAPI, 4 DEG C of chromatography cabinets
5h is incubated in clutch；3500rpm centrifuges 5min, carefully absorbs supernatant；The PBS that 500 μ L contain 1%Triton is often added in pipe clear
Precipitation is washed, is repeated 2 times, finally precipitation is resuspended in 200 μ L PBS；Draw 20 μ L and obtain precipitation, add 4 μ L 6xloading
Buffer boils 5min, and centrifugation draws supernatant and carries out SDS-PAGE electrophoresis.
5. it is according to claim 1 a kind of mutual based on the yellow chicken gene of GST-Pull Down and mass spectral analysis searching Rugao
Make the method for albumen, it is characterized in that, step (6) the protein reductive alkylation and film dosim detailed process are：
The protein after 100 μ gSDS-PAGE electrophoresis is taken out, final concentration 10mM dithiothreitol (DTT)s also crude protein is then added,
Final concentration 55mM iodoacetic acid ammoniums are subsequently added into, 1 μ g trypsase is eventually adding, 8h~16h is digested overnight；It is many that enzymolysis is produced
Peptide C18 pillar desalinations, use loading buffer solution polypeptide, sample-loading buffer active ingredient after the polypeptide of desalination is drained：
0.1% formic acid, 3% acetonitrile, the final concentration of the dissolving of polypeptide is 1 μ g/ μ L.
6. it is according to claim 5 a kind of mutual based on the yellow chicken gene of GST-Pull Down and mass spectral analysis searching Rugao
Make the method for albumen, it is characterized in that, step (6) described LC-MS/MS identifies that detailed process is：
LC-MS/MS (ekspertTM nanoLC on polypeptide；AB Sciex Triple TOF 5600-plus) instrument divided
Analysis, analytical column is AB SCIEX analytical columns, and the specification of AB SCIEX analytical columns is 75 μm of internal diameters, fills 3 μm,'s
ChromXP C18 post material, long 10cm, nozzle needle is NEW objective, and the specification of nozzle needle is 20 μm of internal diameters, the diameter of nozzle needle mouthful
It is 10 μm, trapping column is eksigent Chromxp Trap Column, 3 μm of C18-CL of specification of trapping column, 350μm
×0.5mm。
7. it is according to claim 6 a kind of mutual based on the yellow chicken gene of GST-Pull Down and mass spectral analysis searching Rugao
Make the method for albumen, it is characterized in that, the spectrogram qualification result of the protein digestion polypeptide is shown in Table 1
Machine data analysis parameter under the LC-MS/MS of the polypeptide of table 1
8. it is according to claim 1 a kind of mutual based on the yellow chicken gene of GST-Pull Down and mass spectral analysis searching Rugao
Make the method for albumen, it is characterized in that, different inducing temperatures and isopropylthio gala are designed in step (3) described Induction Process
Glucosides IPTG induced concentration.
9. it is according to claim 1 a kind of mutual based on the yellow chicken gene of GST-Pull Down and mass spectral analysis searching Rugao
Make the method for albumen, it is characterized in that, Pet-49 (b) prokaryotic expression carrier is purchased from BioVector plasmid vector bacterium cells
Gene collection.
10. it is according to claim 1 a kind of mutual based on the yellow chicken gene of GST-Pull Down and mass spectral analysis searching Rugao
Make the method for albumen, it is characterized in that, specific primer passes through Primer5.0 Software for Design in the step (2).
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