CN105777910A - His-Vx3-eGFP protein-nano aluminum covalent coupling ubiquitinated protein containing peptide bonds as well as extraction method and application thereof - Google Patents
His-Vx3-eGFP protein-nano aluminum covalent coupling ubiquitinated protein containing peptide bonds as well as extraction method and application thereof Download PDFInfo
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Abstract
The invention discloses a His-Vx3-eGFP protein-nano aluminum covalent coupling ubiquitinated protein containing peptide bonds as well as an extraction method and application thereof. The ubiquitinated protein is prepared through the following steps of: forming a conjugate of His-Vx3-eGFP protein and modified nano aluminum by virtue of peptide bonds and incubating the conjugate and a treated tumor cell lysis solution. Compared with the prior art, obtained alpha-Al2O3-His-Vx3-eGFP is stable in property, is capable of greatly improving the ubiquitinated protein collection effect and simplifying experiment operation; in addition, the defect that adjuvants are added in use of existing ubiquitinated protein vaccine is overcome.
Description
Technical field
The present invention relates to a kind of albumen of the His-Vx3-eGFP containing peptide bond-nano aluminum covalent coupling ubiquitin protein and carry
Access method, belongs to tumor vaccine technical field.
Background technology
The health of the tumor serious threat mankind.Curative resection operation or chemotherapy, radiotherapy are first-selected the controlling of current tumour patient
Treatment method.Clinical data shows, major part tumour patient is insensitive to the treatment such as chemotherapy, radiotherapy, therefore, and facing of tumor
Bed treatment is in the urgent need to new ways and means.Tumor biotherapy, as a kind of new ideas of cancer therapy, has special
The advantage such as property is strong, toxic and side effects is little, is increasingly paid attention to by people.Wherein, with dendritic cell (DC) swelling as carrier
Tumor vaccine has become the study hotspot of tumor biotherapy the most.
During tumor development, it is the major reason causing immunologic escape that tumor antigen can not effectively be presented to T cell.
Simultaneously in tumor mice, owing to there is the hypofunction even defect of antigen presenting cell, particularly DC is in quantity and function
Change, can cause tumor cell escape body immune surveillance, lead oncogenic formation and development.At present, based on DC
Tumor vaccine is considered as one of maximally effective tumor vaccine, has carried out a large amount of basic research and clinical trial.DC vaccine function
Realization depend primarily on two parts factor, one is that the Effective Antigens of DC offers effect；Two is to load effective antigen, antigen
Information is given full expression to.But, there is many problems in development test has to be solved, including DC source, preparation process,
The antigen load of DC, consumption, usage frequency, immunization route and number of times, and the most preferably raise tumor antigen etc. and become
The focus paid close attention at present.
The antigen polypeptide of pAPC (professional antigen pressing cell, professional antigen deduction cell) intersection submission
It is the material base of inducing specific effector lymphocyte and the antigen polypeptide of the direct submission of tumor cell is that effector lymphocyte effectively knows
The most also target spot of killing tumor cell.On the one hand, the antigen polypeptide of the direct submission of tumor cell is mainly derived from tumor cell
Short-lived albumen (mean half-life is about 10 minutes)；On the other hand, the antigen polypeptide main source of pAPC intersection submission
In the long-lived albumen (degrading through autophagy approach) of tumor cell, there may be asymmetry between the two, therefore, solve
Above-mentioned unsymmetry is the problem that tumour immunity needs to solve.How to make full use of whole antigens letter that tumor cell produces
Breath, the specific effector cell of induction energy more effectively tumor cell is current tumor vaccine institute based on " long-lived albumen "
The difficult problem faced.
It is now recognized that antigen donor's cells mainly has two protein degradation approach: Ubiquitin-Proteasome Pathway and cell autophagy
Approach.Wherein, Ubiquitin-Proteasome Pathway is mainly processed the short-lived albumen of ubiquitination, short-lived albumen by proteasomal degradation
Mainly including the ribosome waste that some error coded, erroneous translation etc. produce, the half-life is only 10 minutes.In recent years
Find, tumor cell MHC I (major histocompatibility complex I, major histocompatibility complex class I)
The antigenic peptides of the direct submission of quasi-molecule has more than 80% to derive from short-lived albumen.Ubiquitin molecule can be with as intracellular signal molecule
The tightest a kind of mode labelled protein molecule.Ubiquitin molecule and target protein or the process of another ubiquitin molecule coupling
Being completed by a series of molecular regulations, these molecules have ubiquitin activating enzyme (E1), ubiquitin cross-linking enzyme (E2) and ubiquitin to connect
Enzyme (E3)；Ubiquitin molecule is finally made to be coupled to the epsilon-amino of polypeptide chain Lys residue or the least significant end group of end amino acid,
Subsequently, another ubiquitin molecule can form ubiquitin chain by being connected with the Lys48 of previous ubiquitin molecule.This connection
The ubiquitin chain of type is the mark as proteasome identification to a great extent.Proteasome is entered by the substrate of ubiquitination
After degraded by proteasome soon.
Early stage we by the gene order of the ubiquitin protein associated proteins (His-Vx3-eGFP) artificial constructed by
Importing escherichia coli (DH5 α) also induces it to express, use method that nickel ion chromatographs with obtain ubiquitin protein and
There is significant antitumous effect.
At present, although prior art has had the research of ubiquitin protein, but, still suffering from problems with needs to solve,
As raised the method complicated difficult of ubiquitin protein to produce in enormous quantities；The ubiquitin protein stable difference of gained is unfavorable for protecting
Deposit；Need during as tumor vaccine to add nano-class aluminum adjuvant.
Summary of the invention
Goal of the invention: in order to solve above-mentioned technical problem, the invention discloses a kind of His-Vx3-eGFP albumen-nano aluminum
Covalent coupling ubiquitin protein and extracting method thereof.
Technical scheme: in order to realize foregoing invention purpose, the invention provides a kind of His-Vx3-eGFP containing peptide bond
Albumen-nano aluminum covalent coupling ubiquitin protein, described ubiquitin protein is by His-Vx3-eGFP albumen with through modifying
After nano aluminum, by peptide bond formed conjugate, then conjugate is hatched with tumor cell lysis liquid after treatment again,
Obtain described ubiquitin protein.
The invention also discloses described His-Vx3-eGFP albumen-nano aluminum covalent coupling ubiquitin protein containing peptide bond
Extracting method, comprises the following steps:
(1) utilize P-hydroxybenzoic acid, nano aluminum granule is modified；
(2) the nano aluminum activation after modifying, adds His-Vx3-eGFP albumen, is formed by generating peptide bond
His-Vx3-eGFP albumen-nano aluminum covalent coupling thing；
(3) culture of tumor cell, the degraded of suppression ubiquitin protein, process through cell pyrolysis liquid, be centrifuged to obtain supernatant；
(4) step (2) gained covalent coupling thing and step (3) gained supernatant are hatched altogether, obtain described ubiquitin
Change protein alpha-Al2O3-His-Vx3-eGFP-Ub。
As preferably, described in step (1), method of modifying is: nano aluminum and P-hydroxybenzoic acid are adding hot oil bath condition
Lower stirring reaction, makes P-hydroxybenzoic acid and the hydroxyl reaction in nano aluminum.
Described nano aluminum in manufacturing process with water generation chemical reaction, formed great amount of hydroxy group.
Preferred as another kind, described in step (2), activation method is: after EDC/NHS modifies with P-hydroxybenzoic acid
Nano aluminum stirring at normal temperature react with activated carboxyl.
Preferred as another kind, described in step (2), His-Vx3-eGFP albumen is to be prepared by following methods:
Recovery has converted the escherichia coli of His-Vx3-eGFP expression plasmid, induces its expressing protein, uses nickel ion layer
Analysis method extracts described His-Vx3-eGFP albumen.
Described His-Vx3-eGFP albumen is Vx3 (A7) fusion protein with ubiquitin binding structural domain, and Vx3 (A7) merges
Albumen derives from Vps27 (a yeast homologue of HRS, Hepatocyte growth for having connected three
Factor-regulated tyrosine kinase substrate) UIM (ubiquitin-interacting motif) artificial constructed
Body, is the artificial constructed egg having very high-affinity to ubiquitin protein being in series with ubiquitin binding structural domain reported in recent years
In vain.
Preferred as another kind, described in step (3), culture of tumor cell is for conventionally to cultivate；
Preferred as another kind, the method suppressing the degraded of ubiquitin protein described in step (3) is: the tumor of cultivation
Cell grows to 80%, adds Bortezomib and ammonium chloride, and addition is respectively (160-240) nmol/L and (25-35) mmol/L,
Intervene tumor cell 14-18 hour, the degraded of suppression ubiquitin protein.
Preferred as another kind, hatching described in step (4) is 4 DEG C of overnight incubation altogether.
Specifically, His-Vx3-eGFP albumen of the present invention-nano aluminum covalent coupling ubiquitin protein, mainly by with
Lower step extraction gained:
(1) modification of nano aluminum granule:
(1a) dry nano aluminum (α-Al is weighed2O3) 100mg, it is placed in 100ml round bottom reactor and adds ultra-pure water
50ml, the hydroxyl in ultrasonic 30 minutes (min) fully activation nano aluminum in ultrasonic cleaning instrument；
(1b) weigh different amounts of P-hydroxybenzoic acid (500,50,5mg) to be separately added in aforesaid reaction vessel, 90 DEG C
Stirring reaction 2h under oil bath；
(1c) by after the above-mentioned reaction abundant washes clean of products therefrom ultra-pure water, it is centrifuged and is precipitated, after lyophilization
Dry potassium bromide (KBr) mixed pressuring plate with appropriate, detects the change of hydroxyl under infrared spectrometer；
(2) His-Vx3-eGFP albumen is covalently coupled to nano aluminum granule:
(2a) recovery has converted the escherichia coli of His-Vx3-eGFP expression plasmid, coats containing 50 μ g/ml ammonia benzyls
On the LB flat board of penicillin, after 14h, picking monoclonal is to 37 DEG C of concussion trainings in the LB culture fluid containing ampicillin
After supporting 6h, draw 1ml and be seeded in 100ml LB culture fluid, treat that it grows to logarithmic (log) phase and adds final concentration 100mM
IPTG low temperature induction 16h；
(2b) obtaining antibacterial by centrifugal for top gained bacterium solution low-temperature and high-speed, PBS washes three times, adds after buffer is resuspended
The lysozyme of 100 μ g/ml acts on 20min after acting on 20min on ice on sonicator, and high-speed low temperature is centrifugal to be obtained
Supernatant；
(2c) sway reaction overnight by above-mentioned supernatant addition nickel ion affinity chromatograph post 4 DEG C, within second day, use Washing
Buffer washes away foreign protein, Elution buffer eluting His-Vx3-eGFP albumen；
(2d) with EDC/NHS, the product activation in (1c), PBS are added step (2c) gained after washing three times
Overnight, second day low-temperature and high-speed is centrifuged collects nano aluminum and His-Vx3-eGFP to the stirring reaction of 4 DEG C of His-Vx3-eGFP albumen
Covalent coupling product (α-the Al of albumen2O3-His-Vx3-eGFP)；
(3) cultivation of tumor cell:
Recovering frozen 4T1 cell, 40 DEG C of water-baths add containing 10% (V/V) hyclone, 100U/ml blue or green immediately after melting
The RPMI-1640 culture medium of mycin and 100U/ml streptomycin, re-suspended cell to 1 × 106Individual/ml, and move it into 500ml
In Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO2Constant incubator in cultivate, within every 1～2 day, change liquid once, conventional
0.25% (g/mL) trypsinization passes on；
(4) process of tumor cell:
Cultivating cell by step (3) method and treat that cell grows to 80%, Bortezomib and ammonium chloride, addition is respectively (160-240)
Nmol/L and (25-35) mmol/L, intervenes tumor cell 14-18 hour, the degraded of suppression ubiquitin protein；By institute
Cell after cell pyrolysis liquid processes, centrifugal obtain supernatant；
(5) ubiquitin protein is raised:
Nano aluminum granule 4 DEG C after step (4) gained cell pyrolysis liquid and the covalent coupling of step (2) gained is incubated altogether
Educating overnight, second day low-temperature and high-speed is centrifuged the connection product obtaining ubiquitin protein with nano aluminum
(α-Al2O3-His-Vx3-eGFP-Ub)；
(6) ubiquitin protein that western blot detection is raised:
By step (5) gained α-Al2O3-His-Vx3-eGFP-Ub is resuspended with appropriate PBS, draw 100 μ l at a high speed from
Supernatant discarded after the heart, adds 100 μ l SDS-PAGE loading buffer, boiling water bath 5min, centrifuging and taking supernatant
After SDS-PAGE, add anti-ub antibody and K63 antibody test gained Ub albumen；
The present invention finally also discloses described His-Vx3-eGFP albumen-nano aluminum covalent coupling ubiquitin protein in preparation
Application in anti-tumor vaccine.Described His-Vx3-eGFP albumen-nano aluminum covalent coupling ubiquitin protein can effectively press down
Growth of tumour cell processed, extends the time-to-live of lotus tumor live body.
Technique effect: relative to prior art, the invention have the advantages that
1, the present invention substantial amounts of can raise ubiquitin protein, and yield to vaccine is greatly improved.
2, gained α-Al of the present invention2O3-His-Vx3-eGFP stable in properties can be stored for a long time, it is possible to from different tumor cells
Lysate is conveniently raised ubiquitin protein.
3, the vaccine of gained of the present invention is directly with nano-class aluminum adjuvant coupling, without extra interpolation adjuvant during use.
Accompanying drawing explanation
Fig. 1: infrared spectrum detection nano aluminum is with the reaction of P-hydroxybenzoic acid；
Fig. 2: confocal laser scanning microscope α-Al2O3Specific covalent coupling His-Vx3-eGFP albumen；
Fig. 3: laser co-focusing observes α-Al2O3The stability of-His-Vx3-eGFP；
Fig. 4:. the detection of drug treating different time 4T1 cytoactive；
Fig. 5:. the expression of drug treating different time 4T1 cellular ubiquitination albumen；
Fig. 6: different pharmaceutical processes the situation of 4T1 cellular ubiquitination protein expression；
Fig. 7: A. α-Al2O3-His-Vx3-eGFP raises ubiquitin protein and raises the detection method of rear albumen；B.western
Blot detects α-Al2O3The ubiquitin protein that-His-Vx3-eGFP raises.
Detailed description of the invention
Further describe the technical solution of the present invention below in conjunction with the accompanying drawings.
Embodiment 1: the modification of nano aluminum granule
1. weigh dry nano aluminum (α-Al2O3) 100mg, it is placed in 100ml round bottom reactor and adds ultra-pure water 50ml,
In ultrasonic cleaning instrument, ultrasonic 30min fully activates the hydroxyl in nano aluminum；
2. weigh different amounts of P-hydroxybenzoic acid (500,50,5mg) to be separately added in aforesaid reaction vessel, 90 DEG C of oil
Bath lower stirring reaction 2h；
3. by after the above-mentioned reaction abundant washes clean of products therefrom ultra-pure water, it is centrifuged and is precipitated, with suitable after lyophilization
Dry potassium bromide (KBr) mixed pressuring plate of amount, detects the change of hydroxyl under infrared spectrometer；
As shown in Figure 1: the hydroxyl peak on nano aluminum granule is very strong, along with the increase nanometer with P-hydroxybenzoic acid response magnitude
Hydroxyl peak on aluminum gradually weakens, it was demonstrated that P-hydroxybenzoic acid is successfully connected to nano aluminum and along with P-hydroxybenzoic acid
The increase of amount, the P-hydroxybenzoic acid that nano aluminum combines gradually increases.
Embodiment 2:His-Vx3-eGFP albumen is covalently coupled to nano aluminum granule
1. recovery has converted the escherichia coli of His-Vx3-eGFP expression treatment, coats containing 50 μ g/ml ammonia benzyl penicillium sp
On the LB flat board of element, after 14h, picking monoclonal cultivates 6h to 37 DEG C of concussions in the LB culture fluid containing ampicillin
After, draw 1ml and be seeded in 100ml LB culture fluid, treat that it grows to the IPTG that logarithmic (log) phase adds final concentration 100mM
Low temperature induction 16h；
2. obtaining antibacterial by centrifugal for top gained bacterium solution low-temperature and high-speed, PBS washes three times, adds after buffer is resuspended
The lysozyme of 100 μ g/ml acts on 20min after acting on 20min on ice on sonicator, and high-speed low temperature is centrifugal to be obtained
Supernatant；
3. sway reaction overnight by above-mentioned supernatant addition nickel ion affinity chromatograph post 4 DEG C, within second day, use Washing buffer
Wash away foreign protein, Elution buffer eluting His-Vx3-eGFP albumen；
4. with EDC/NHS, the product activation in (1c), PBS are added step (2c) gained after washing three times
Overnight, second day low-temperature and high-speed is centrifuged collects nano aluminum and His-Vx3-eGFP to the stirring reaction of 4 DEG C of His-Vx3-eGFP albumen
Covalent coupling product (α-the Al of albumen2O3-His-Vx3-eGFP)；
Embodiment 3: the specificity of nano aluminum covalent coupling His-Vx3-eGFP albumen
1. nano aluminum is after P-hydroxybenzoic acid is modified, without EDC/NHS catalysis directly with His-Vx3-eGFP albumen
Hatch altogether with the mouse IgG antibody of R-PE labelling again after hatching altogether.As in figure 2 it is shown, under laser confocal microscope
Observe: the aluminium oxide after P-hydroxybenzoic acid is modified can also can same mouse IgG in conjunction with His-Vx3-eGFP albumen
In conjunction with, it was demonstrated that the nano aluminum after modification can be in conjunction with arbitrary protein.
2. nano aluminum is after P-hydroxybenzoic acid is modified, and is catalyzed with EDC/NHS, then with His-Vx3-eGFP albumen
Hatching altogether, after reaction terminates, PBS washes the mouse IgG antibody adding P-PE labelling three times hatches altogether.Such as Fig. 2 institute
Show, observe under laser confocal microscope: the nano aluminum after P-hydroxybenzoic acid is modified is catalyzed through EDC/NHS
After can with His-Vx3-eGFP albumen coupling, but the product after coupling still has the ability combining other albumen.
3. nano aluminum is after P-hydroxybenzoic acid is modified, then modifies with n-butyl alcohol, the most same without EDC/NHS catalysis
His-Vx3-eGFP albumen is hatched with the mouse IgG antibody of R-PE labelling after hatching altogether more altogether.As in figure 2 it is shown, swashing
Observe under light Laser Scanning Confocal Microscope: the nano aluminum after n-butyl alcohol is modified can not be in conjunction with any albumen.
4. nano aluminum is after P-hydroxybenzoic acid is modified, then modifies with n-butyl alcohol, after EDC/NHS is catalyzed, with
His-Vx3-eGFP albumino reaction, PBS adds the mouse IgG antibody of R-PE labelling and hatches altogether after washing three times.Such as Fig. 2
Shown in, observe under laser confocal microscope: His-Vx3-eGFP albumen can be covalently bond to nano aluminum, and
And product can not greatly reduce in conjunction with the amount of other albumen.
5. nano aluminum is after P-hydroxybenzoic acid is modified, then modifies with n-butyl alcohol, after EDC/NHS is catalyzed, with
His-Vx3-eGFP albumino reaction, PBS adds 5%BSA after washing three times and hatches 2h altogether, then little with R-PE labelling
Mus IgG antibody is hatched altogether.As in figure 2 it is shown, observe under laser confocal microscope: His-Vx3-eGFP albumen
Nano aluminum can be covalently bond to, and can not be in conjunction with other albumen through BSA effect afterproduct.
Embodiment 4: the research of nano aluminum covalent coupling His-Vx3-eGFP protein product stability
1. by the α-Al after covalent coupling2O3-His-Vx3-eGFP is placed in 4 DEG C, stands 3 weeks.Laser confocal microscope
Observe, such as Fig. 3 (a): the product of covalent coupling still has the strongest fluorescence after standing 3 weeks, it was demonstrated that
α-Al2O3-His-Vx3-eGFP has good stability.
2. by the mixture (α-Al of nano aluminum Yu His-Vx3-eGFP albumen2O3+ His-Vx3-eGFP) it is placed in 4 DEG C,
Stand 3 weeks.Confocal laser scanning microscope, such as Fig. 3 (b): nano aluminum is just mixed with His-Vx3-eGFP albumen
During conjunction, fluorescence intensity is the highest, but is substantially not visible fluorescence after standing 3 weeks, it was demonstrated that α-Al2O3-His-Vx3-eGFP is with receiving
Rice aluminum and the mixture (α-Al of His-Vx3-eGFP albumen2O3+ His-Vx3-eGFP) compare good stability.
Embodiment 5:4T1 cell expresses the condition that ubiquitin protein is the highest
1. the cultivation of tumor cell:
Recovering frozen 4T1 cell (mouse mastopathy cell), 40 DEG C of water-baths add immediately containing 10% (V/V) tire cattle after melting
The RPMI-1640 culture medium of serum, 100U/ml penicillin and 100U/ml streptomycin, re-suspended cell to 1 × 106Individual/ml,
And move it in 500ml Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO2Constant incubator in cultivate, every 1～2
It changes liquid once, and conventional 0.25% (g/mL) trypsinization passes on；
2. the process of tumor cell:
1) cultivate cell by step 1 method and treat that cell grows to 80%, Bortezomib and ammonium chloride, addition 100nmol/L
And 20mmol/L, intervene tumor cell 3,6,9,12 hours, the degraded of suppression ubiquitin protein；CCK-8 method is examined
Survey the activity of cell.As shown in Figure 4: along with the activity increasing cell of action time is increasingly suppressed, act on 12
H cytoactive only has 80%.
2) cultivate cell by step 1 method and treat that cell grows to 80%, Bortezomib and ammonium chloride, addition 100nmol/L
And 20mmol/L, intervene tumor cell 3,6,9,12 hours, the degraded of suppression ubiquitin protein；Collection cell adds
Enter RIPA cell pyrolysis liquid and act on 20min, collected after centrifugation supernatant on ice, take appropriate supernatant and add SDS-PAGE
Loading buffer, goes to pvdf membrane by albumen after electrophoresis, adds antibody and the antibody of anti-K63ub of anti-ub.4℃
Overnight incubation, within second day, PBST washes film 5 times, each 5min, adds the two of corresponding HRP labelling and resists, and room temperature is incubated
Educate 1h, PBST cell 5 times, each 5min, add colour reagent and take pictures.As it is shown in figure 5, western blot bar
Band shows, medicine effect 9h, and the expression of intracellular total ub and K63ub is the highest.
β-tubilin
3) cultivate cell by step 1 method and treat that cell grows to 80%, be separately added into Bortezomib 100nmol/L or ammonium chloride
20mmol/L, intervenes tumor cell 9h, the degraded of suppression ubiquitin protein；Collect cell and add the cracking of RIPA cell
Liquid acts on 20min, collected after centrifugation supernatant on ice, takes appropriate supernatant and adds SDS-PAGE loading buffer, electrophoresis
After albumen is gone to pvdf membrane, add antibody and the antibody of anti-K63ub of anti-ub.4 DEG C of overnight incubation, second day
PBST washes film 5 times, each 5min, adds the two of corresponding HRP labelling and resists, incubated at room 1h, PBST cell 5
Secondary, each 5min, add colour reagent and take pictures.As shown in Figure 6, western blot band shows, two kinds of medicines are altogether
Same-action 9h, the expression of intracellular total ub and K63ub is the highest.
Embodiment 6: the His-Vx3-eGFP albumen being coupled to nano aluminum raises ubiquitin protein from cell pyrolysis liquid
1. the nano aluminum granule 4 DEG C after embodiment 5 gained cell pyrolysis liquid and the covalent coupling of embodiment 1 gained is incubated altogether
Educating overnight, second day low-temperature and high-speed is centrifuged the connection product obtaining ubiquitin protein with nano aluminum
(α-Al2O3-His-Vx3-eGFP-Ub)。
2. by step 1 gained α-Al2O3-His-Vx3-eGFP-Ub is resuspended with appropriate PBS, draws 100 μ l high speed centrifugations
Rear supernatant discarded, adds 100 μ l SDS-PAGE sample-loading buffer (loading buffer), boiling water bath 5min, centrifugal
After taking supernatant SDS-PAGE, add anti-ub antibody and K63 antibody test gained Ub albumen；Result as it is shown in fig. 7,
α-Al2O3-His-Vx3-eGFP can raise substantial amounts of ubiquitin protein.
Claims (9)
1. the His-Vx3-eGFP albumen-nano aluminum covalent coupling ubiquitin protein containing peptide bond, it is characterized in that, described ubiquitin protein is by His-Vx3-eGFP albumen and the nano aluminum after modifying, conjugate is formed by peptide bond, then conjugate is hatched with tumor cell lysis liquid after treatment again, obtains described ubiquitin protein.
2. the extracting method of the albumen of the His-Vx3-eGFP containing peptide bond described in claim 1-nano aluminum covalent coupling ubiquitin protein, it is characterised in that comprise the following steps:
(1) utilize P-hydroxybenzoic acid, nano aluminum granule is modified；
(2) the nano aluminum activation after modifying, adds His-Vx3-eGFP albumen, forms His-Vx3-eGFP albumen-nano aluminum covalent coupling thing by generating peptide bond；
(3) culture of tumor cell, the degraded of suppression ubiquitin protein, process through cell pyrolysis liquid, be centrifuged to obtain supernatant；
(4) step (2) gained covalent coupling thing and step (3) gained supernatant are hatched altogether, obtain described ubiquitin protein α-Al2O3-His-Vx3-eGFP-Ub。
Extracting method the most according to claim 2, it is characterised in that described in step (1), method of modifying is: nano aluminum and P-hydroxybenzoic acid stir reaction under the conditions of adding hot oil bath, makes P-hydroxybenzoic acid and the hydroxyl reaction in nano aluminum.
Nano aluminum stirring at normal temperature after extracting method the most according to claim 2, it is characterised in that described in step (2), activation method is: EDC/NHS modifies with P-hydroxybenzoic acid is reacted with activated carboxyl.
Extracting method the most according to claim 2, it is characterised in that described in step (2), His-Vx3-eGFP albumen is to be prepared by following methods:
Recovery has converted the escherichia coli of His-Vx3-eGFP expression plasmid, induces its expressing protein, uses nickel ion chromatography to extract described His-Vx3-eGFP albumen.
Extracting method the most according to claim 2, it is characterised in that described in step (3), culture of tumor cell is for conventionally to cultivate.
Extracting method the most according to claim 2, it is characterized in that, the method suppressing the degraded of ubiquitin protein described in step (3) is: the growth of tumour cell of cultivation to 80%, add Bortezomib and ammonium chloride, addition is respectively (160-240) nmol/L and (25-35) mmol/L, intervene tumor cell 14-18 hour, the degraded of suppression ubiquitin protein.
Extracting method the most according to claim 2, it is characterised in that hatching described in step (4) is 4 DEG C of overnight incubation altogether.
9. the His-Vx3-eGFP albumen described in claim 1-nano aluminum covalent coupling ubiquitin protein application in preparing anti-tumor vaccine.
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| CN110669111A (en) * | 2019-10-12 | 2020-01-10 | 东南大学 | Cancer stem cell-like drug-resistant cell-derived ubiquitinated protein and application thereof in preparation of anti-cancer drugs |
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| CN104877017A (en) * | 2015-05-06 | 2015-09-02 | 东南大学 | Anti-tumor ubiquitinated protein and extraction method and application thereof |
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| CN104877017A (en) * | 2015-05-06 | 2015-09-02 | 东南大学 | Anti-tumor ubiquitinated protein and extraction method and application thereof |
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| MOHANAD ALDAROUISH ET AL.: "Ubiquitinated proteins enriched from tumor cells by a ubiquitin binding protein Vx3(A7) as a potent cancer vaccine.", 《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》 * |
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| CN110669111A (en) * | 2019-10-12 | 2020-01-10 | 东南大学 | Cancer stem cell-like drug-resistant cell-derived ubiquitinated protein and application thereof in preparation of anti-cancer drugs |
| CN110669111B (en) * | 2019-10-12 | 2021-07-09 | 东南大学 | Cancer stem cell-like drug-resistant cell-derived ubiquitinated protein and application thereof in preparation of anti-cancer drugs |
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