CN104877017A - Anti-tumor ubiquitinated protein and extraction method and application thereof - Google Patents
Anti-tumor ubiquitinated protein and extraction method and application thereof Download PDFInfo
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Abstract
The invention discloses anti-tumor ubiquitinated protein which is mainly extracted from the following steps: (1) culture of tumor cells: resuscitating cryopreserved tumor cells and culturing the tumor cells according to a conventional culture method; (2) treatment of the tumor cells; and (3) extraction of the ubiquitinated protein. The invention further discloses an extraction method of the anti-tumor ubiquitinated protein as well as application of the anti-tumor ubiquitinated protein in preparing tumor therapeutic vaccines. Compared to the prior art, the protein disclosed by the invention can be used for greatly improving the effect of cross-immuno reaction and improving the inhibitory effect on different types of tumors to a great extent; in addition, the problem that ubiquitinated short-lived protein is fast to degrade is further overcome, and a lot of ubiquitinated protein can be quickly obtained. Finally, the asymmetry between the short-lived protein and the long-lived protein is overcome, the target spots for inducing specific effector cells are increased, and the defect that the anti-tumor vaccines are poor in anti-tumor effect based on the long-lived protein is remedied.
Description
Technical field
The present invention relates to a kind of ubiquitin protein and extracting method thereof and application, belong to tumor vaccine technical field.
Background technology
The health of the tumour serious threat mankind.Healing property resection operation or chemotherapy, radiotherapy are the methods for the treatment of of current tumour patient first-selection.Clinical data shows, and most of tumour patient is insensitive to the treatment such as chemotherapy, radiotherapy, and therefore, the clinical treatment of tumour is in the urgent need to new ways and means.Tumor biotherapy, as a kind of new ideas of cancer therapy, has the advantages such as high specificity, toxic side effect are little, is more and more subject to people and payes attention to.Wherein, the tumor vaccine being carrier with dendritic cell (DC) has become the study hotspot of tumor biotherapy at present.
In tumor development process, tumour antigen can not be effectively the major reason causing immunologic escape in passing T cell.Simultaneously in tumor mice, because the hypofunction even defect, particularly DC that there is antigen presenting cell is in the change of quantity and function, tumour cell can be caused to escape the immunosurveillance of body, lead oncogenic formation and development.At present, the tumor vaccine based on DC is considered to one of the most effective tumor vaccine, has carried out a large amount of fundamental research and clinical trial.The realization of DC vaccine function depends primarily on two portions factor, and one is the effect of offering of the Effective Antigens of DC; Two is the effective antigen of load, and antigenic information is given full expression to.But there is many problems in research trial has to be solved, comprise DC source, preparation process, the antigen load of DC, consumption, usage frequency, immunization route and number of times, and how better to raise tumour antigen etc. and become the focus paid close attention at present.
PAPC (professional antigen pressing cell, professional antigen deduction cell) intersects the antigenic peptide of submission, and be the basic substance of inducing specific effector cell and the antigenic peptide of the direct submission of tumour cell to be effector cell effectively identify and the target spot of killing tumor cell.On the one hand, the antigenic peptide of the direct submission of tumour cell is mainly derived from the short-lived albumen (mean half-life is about 10 minutes) of tumour cell; On the other hand, the antigenic peptide of pAPC intersection submission is mainly derived from the long-lived albumen (through the degraded of autophagy approach) of tumour cell, may there is asymmetry between the two, therefore, solving above-mentioned asymmetry is the problem that tumour immunity needs to solve.How to make full use of whole antigenic informations that tumour cell produces, induction can the specific effector cell of more effective tumor cell be the difficult problem that the tumor vaccine at present based on " long-lived albumen " faces.
Think that antigen donor's cells mainly contains two proteolytic degradation approach at present: Ubiquitin-Proteasome Pathway and cell autophagy approach.Wherein, Ubiquitin-Proteasome Pathway is primarily of the short-lived albumen of proteasomal degradation process ubiquitination, and short-lived albumen mainly comprises the rrna waste prods that some error coded, erroneous translation etc. produce, and the transformation period is only 10 minutes.Discovered in recent years, the antigen peptide of tumour cell MHC I (major histocompatibility complex I, major histocompatibility complex class I) the direct submission of quasi-molecule has more than 80% to derive from short-lived albumen.Ubiquitin molecule as intracellular signal molecular energy with a kind of extremely tight mode labelled protein molecule.The process of ubiquitin molecule and target protein or another ubiquitin molecule coupling is completed by a series of molecular regulation, and these molecules have ubiquitin activating enzyme (E1), ubiquitin crosslinking enzyme (E2) and ubiquitin ligase (E3); Finally make ubiquitin molecule 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.The ubiquitin chain of this connection type is the mark as proteasome identification to a great extent.After being entered proteasome by the substrate of ubiquitination soon degrade by proteasome.
Therefore, there is following problem in prior art needs to solve, and as cross immunity weak effect, the short-lived proteolytic degradation of ubiquitination is fast, is difficult to extract; The short-lived albumen of ubiquitination and long-lived albumen have asymmetry, cause the tumor vaccine antitumous effect based on " long-lived albumen " not good.
Summary of the invention
Goal of the invention: in order to solve the problems of the technologies described above, the invention discloses a kind of antitumor ubiquitin protein and extracting method thereof and application.
Technical scheme: in order to realize foregoing invention object, the invention provides a kind of antitumor ubiquitin protein, it mainly extracts gained by following steps:
(1) cultivation of tumour cell: frozen tumour cell of recovering, conveniently cultural method is cultivated;
(2) process of tumour cell:
Growth of Cells to 80% is treated by step (1) method culturing cell, add rapamycin, Bortezomib (Velcade, commodity are called Bortezomib) and ammonium chloride, add-on is respectively (80-120) nmol/L, (160-240) nmol/L and (25-35) mmol/L, intervene tumour cell 14-18 hour, suppress the degraded of ubiquitin protein;
(3) ubiquitin protein is extracted:
By step (2) gained cell through cell pyrolysis liquid (Western and IP cell pyrolysis liquid, the green skies, Jiangsu) process after, centrifugally to be precipitated, to get supernatant liquor and same Vx3 (A7) albumen is hatched altogether, can ubiquitin protein be obtained through nickel ion affinity chromatograph.
As preferably, described tumour cell is multiple myeloma cells or melanoma cell.
Further preferably, described multiple myeloma cells is mouse EL4 cell, and described melanoma cell is mouse B16-F10 cell.
As preferably, the conventional culture methods in described step (1) is as follows:
To recover frozen tumour cell, 40 DEG C of water-baths add DMEM (the Dulbecco's Modified Eagle's Medium) substratum containing 10% (v/v) foetal calf serum, 100U/ml penicillin and 100U/ml Streptomycin sulphate immediately after melting, re-suspended cell to 1 × 10
6individual/ml, and moved in Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO
2constant incubator in cultivate, within every 1 ~ 2 day, change liquid once, conventional 0.25% tryptic digestion goes down to posterity;
As preferably, the extracting method of described Vx3 (A7) albumen comprises the steps:
(1) pUbiG101-Vx3 (A7)-eGFP plasmid is proceeded to E. coli (DH5-α);
(2) intestinal bacteria picking mono-clonal step (1) obtained, LB (Luria-Bertani) culture medium culturing also does double digestion qualification;
(3) the bacterium enlarged culturing after step (2) being identified, IPTG (Isopropyl β-D-1-thiogalactopyranoside is added when light absorption value is 0.5 under bacterium liquid 600nm wavelength, isopropyl-β-D-thiogalactoside(IPTG)) induce 16h, high speed centrifugation 8000rpm, 15min, supernatant discarded;
(4) precipitation Bing buffer (the 50mM NaH will obtained after step (3) high speed centrifugation
2pO
4, 300mMNaCl, 25mM imidazole, pH8.0) and resuspended, add N,O-Diacetylmuramidase and react 30min on ice; After ultrasonication, high speed centrifugation 12000rpm, 10min, 4 DEG C;
(5) collect the supernatant that step (4) obtains, joined in nickel ion affinity chromatograph, after being combined with column material, with Washing buffer (50mM NaH
2pO4,300mM NaCl, 150mM imidazole, pH8.0) wash away foreign protein, with Elution buffer (50mM NaH
2pO4,300mM NaCl, 300mM imidazole, pH8.0) collect desired Vx3 (A7) albumen, frozen in-20 DEG C, for subsequent use.
Present invention also offers the extracting method of described antitumor ubiquitin protein, it comprises the following steps:
(1) cultivation of tumour cell: frozen tumour cell of recovering, conveniently cultural method is cultivated;
(2) process of tumour cell:
Growth of Cells to 80% is treated by step (1) method culturing cell, add rapamycin, Bortezomib and ammonium chloride, add-on is respectively (80-120) nmol/L, (160-240) nmol/L and (25-35) mmol/L, intervene tumour cell 14-18 hour, suppress the degraded of ubiquitin protein;
(3) ubiquitin protein is extracted:
By step (2) gained cell after cell pyrolysis liquid process, to be centrifugally precipitated, to get supernatant liquor and same Vx3 (A7) albumen is hatched altogether, can ubiquitin protein be obtained through nickel ion affinity chromatograph.
As preferably, described tumour cell is multiple myeloma cells or melanoma cell.
Further preferably, described multiple myeloma cells is mouse EL4 cell, and described melanoma cell is mouse B16-F10 cell.
Preferred as another kind, the conventional culture methods in described step (1) is as follows:
To recover frozen tumour cell, 40 DEG C of water-baths add the DMEM substratum containing 10% (v/v) foetal calf serum, 100U/ml penicillin and 100U/ml Streptomycin sulphate immediately after melting, re-suspended cell to 1 × 106/ml, and moved in Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO
2constant incubator in cultivate, within every 1 ~ 2 day, change liquid once, conventional 0.25% tryptic digestion goes down to posterity.
Preferred as another kind, the extracting method of described Vx3 (A7) albumen comprises the steps:
(1) pUbiG101-Vx3 (A7)-eGFP plasmid is proceeded to E. coli (DH5-α);
(2) intestinal bacteria picking mono-clonal step (1) obtained, LB culture medium culturing also does double digestion qualification;
(3) the bacterium enlarged culturing after step (2) being identified, adds IPTG and induces 16h, high speed centrifugation 8000rpm, 15min, supernatant discarded when light absorption value is 0.5 under bacterium liquid 600nm wavelength;
(4) by resuspended for the precipitation Bing buffer obtained after step (3) high speed centrifugation, add N,O-Diacetylmuramidase and react 30min on ice; After ultrasonication, high speed centrifugation 12000rpm, 10min, 4 DEG C;
(5) collect the supernatant that step (4) obtains, joined in nickel ion affinity chromatograph, after being combined with column material, foreign protein is washed away with Washing buffer, desired Vx3 (A7) albumen is collected with Elution buffer, frozen in-20 DEG C, for subsequent use.
The present invention finally also provides described antitumor ubiquitin protein preparing the application in tumor therapeutic vaccine.
As preferably, described vaccine is combined by ubiquitin protein and nano-class aluminum adjuvant to make.
The present invention is by after blocking-up Cellular proteasomes approach, with the associated protein body with ubiquitin combined function, these are had the short-lived albumen of the specific ubiquitination of potential tumor to separate from cell pyrolysis liquid, the vaccine that then associating nano-class aluminum adjuvant is made causes specific immune response.
The albumen with ubiquitin binding domains that the present invention uses is Vx3 (A7) fusion rotein, Vx3 (A7) fusion rotein derives from Vps27 (a yeast homologue of HRS for having connected three, Hepatocyte growth factor-regulatedtyrosine kinase substrate) the artificial construct of UIM (ubiquitin-interacting motif), be artificial constructed albumen ubiquitin protein being had to very high-affinity being in series with ubiquitin binding domains reported in recent years.
The experimental results shows: adopt autophagy inhibitor (3-AM, wortmannin), siRNA technology to lower the autophagy of the method inhibition tumor cells such as autophagy related gene (Atg12), then obviously reduce the immune effect that DC intersection offers tumour antigen; Otherwise, adopt autophagy inductor (rapamycin, NH
4cL), the method inducing tumor cell such as hungry produces cell autophagy, then obviously strengthen DC and intersect the immune effect of offering.Containing a large amount of ubiquitin proteins in cytolysosome, therefore with Vx3 (A7) fusion rotein, the ubiquitin protein in tumour cell can be extracted.
Technique effect: relative to prior art, the present invention has the following advantages:
1, albumen of the present invention can improve the effect of cross-immune reaction greatly, significantly improves the inhibition to different sorts tumour.
2, a large amount of antitumor ubiquitin protein of quick obtaining.The present invention adopts 3 kinds of Drug combination process tumour cells, suppresses the degraded of ubiquitin protein, from cells and supernatant, then extracts the ubiquitin protein of tumour cell using Vx3 (A7) fusion rotein as carrier.
3, short-lived for ubiquitination protein extraction is out made vaccine by the present invention, the asymmetry between short-lived albumen and long-lived albumen can be overcome, increase the target spot of inducing specific effector cell, make up the defect that tumor vaccine antitumous effect based on " long-lived albumen " is not good.
4, the present invention utilize nano-class aluminum adjuvant can effectively inducing mouse INF-γ generation and the growth of mouse interior tumor cell can be suppressed.
5, ubiquitin protein of the present invention is as a kind of novel tumor vaccine, immune mouse, can suppress the growth of mouse tumor in vivo.
Accompanying drawing explanation
Ubiquitin protein detected result in Fig. 1: A.EL4 cell lysate; Ubiquitin protein detected result in B.B16-F10 cell lysate;
Fig. 2: the immune response result of various dose ubiquitin protein induction;
Fig. 3: the immune response result of ubiquitin protein induction;
Fig. 4: the immune response results contrast of different immunization ways;
Fig. 5: the knurl specific immune response result (ubiquitin protein of EL4 cell derived) that ubiquitin protein and DRibble induce;
Fig. 6: the knurl specific immune response result (ubiquitin protein of B16-F10 cell derived) that ubiquitin protein and DRibble induce;
Fig. 7: the knurl specific immunity cross reaction result (embodiment 5 gained ubiquitin protein) of ubiquitin protein induction;
Fig. 8: the knurl specific immunity cross reaction result (embodiment 6 gained ubiquitin protein) of ubiquitin protein induction;
Fig. 9: the knurl specific immunity cross reaction result (embodiment 3 gained ubiquitin protein) of ubiquitin protein induction;
Figure 10: the knurl specific immunity cross reaction result (embodiment 4 gained ubiquitin protein) of ubiquitin protein induction;
The mouse interior tumor size variation situation of Figure 11: EL4 ubiquitin protein immunity;
The mouse survival situation of Figure 12: EL4 ubiquitin protein immunity;
Figure 13: the mouse interior tumor size variation situation (mouse EL4 tumor model) (embodiment 5 and embodiment 6 gained ubiquitin protein) of different ubiquitin protein immunity;
Figure 14: the mouse survival situation (mouse EL4 tumor model) (embodiment 5 and embodiment 6 gained ubiquitin protein) of different ubiquitin protein immunity;
Figure 15: the mouse interior tumor size variation situation (mouse B16-F10 tumor model) (embodiment 5 and embodiment 6 gained ubiquitin protein) of different ubiquitin protein immunity;
Figure 16: the mouse survival situation (mouse B16-F10 tumor model) (embodiment 5 and embodiment 6 gained ubiquitin protein) of different ubiquitin protein immunity.
Figure 17: the mouse interior tumor size variation situation (mouse EL4 tumor model) (embodiment 3 and embodiment 4 gained ubiquitin protein) of different ubiquitin protein immunity;
Figure 18: the mouse survival situation (mouse EL4 tumor model) (embodiment 3 and embodiment 4 gained ubiquitin protein) of different ubiquitin protein immunity;
Figure 19: the mouse interior tumor size variation situation (mouse B16-F10 tumor model) (embodiment 3 and embodiment 4 gained ubiquitin protein) of different ubiquitin protein immunity;
Figure 20: the mouse survival situation (mouse B16-F10 tumor model) (embodiment 3 and embodiment 4 gained ubiquitin protein) of different ubiquitin protein immunity.
Embodiment
Technical solution of the present invention is further described below in conjunction with accompanying drawing.
Embodiment 1:
One, the extracting method of Vx3 (A7) albumen:
(1) pUbiG101-Vx3 (A7)-eGFP plasmid is proceeded to E. coli (DH5-α);
(2) intestinal bacteria picking mono-clonal step (1) obtained, LB (Luria-Bertani) culture medium culturing also does double digestion qualification;
(3) the bacterium enlarged culturing after step (2) being identified, IPTG (Isopropyl β-D-1-thiogalactopyranoside is added when light absorption value is 0.5 under bacterium liquid 600nm wavelength, isopropyl-β-D-thiogalactoside(IPTG)) induce 16h, high speed centrifugation 8000rpm, 15min, supernatant discarded;
(4) precipitation Bing buffer (the 50mM NaH will obtained after step (3) high speed centrifugation
2pO
4, 300mMNaCl, 25mM imidazole, pH8.0) and resuspended, add N,O-Diacetylmuramidase and react 30min on ice; After ultrasonication, high speed centrifugation 12000rpm, 10min, 4 DEG C;
(5) collect the supernatant that step (4) obtains, joined in nickel ion affinity chromatograph, after being combined with column material, with Washing buffer (50mM NaH
2pO4,300mM NaCl, 150mM imidazole, pH8.0) wash away foreign protein, with Elution buffer (50mM NaH
2pO4,300mM NaCl, 300mM imidazole, pH8.0) collect desired Vx3 (A7) albumen, frozen in-20 DEG C, for subsequent use.
Two, the extracting method of ubiquitin protein:
(1) cultivation of tumour cell: the EL4 cell of recovering frozen, 40 DEG C of water-baths add the DMEM substratum containing 10% (v/v) foetal calf serum, 100U/ml penicillin and 100U/ml Streptomycin sulphate immediately after melting, re-suspended cell to 1 × 10
6individual/ml, and moved in 500ml Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO
2constant incubator in cultivate, within every 1 ~ 2 day, change liquid once, conventional 0.25% tryptic digestion goes down to posterity;
(2) process of tumour cell:
Cultivate by step (1) method, treat Growth of Cells to 80%, add rapamycin, Bortezomib and ammonium chloride, add-on is respectively 80nmol/L, 160nmol/L and 25mmol/L, intervenes tumour cell 14 hours, suppresses the degraded of ubiquitin protein;
(3) ubiquitin protein is extracted:
By step (2) gained cell after cell pyrolysis liquid process, to be centrifugally precipitated, to get supernatant liquor and same Vx3 (A7) albumen is hatched altogether, can ubiquitin protein be obtained through nickel ion affinity chromatograph.
Embodiment 2:
One, the extracting method of Vx3 (A7) albumen:
(1) pUbiG101-Vx3 (A7)-eGFP plasmid is proceeded to E. coli (DH5-α);
(2) intestinal bacteria picking mono-clonal step (1) obtained, LB (Luria-Bertani) culture medium culturing also does double digestion qualification;
(3) the bacterium enlarged culturing after step (2) being identified, IPTG (Isopropyl β-D-1-thiogalactopyranoside is added when light absorption value is 0.5 under bacterium liquid 600nm wavelength, isopropyl-β-D-thiogalactoside(IPTG)) induce 16h, high speed centrifugation 8000rpm, 15min, supernatant discarded;
(4) precipitation Bing buffer (the 50mM NaH will obtained after step (3) high speed centrifugation
2pO
4, 300mMNaCl, 25mM imidazole, pH8.0) and resuspended, add N,O-Diacetylmuramidase and react 30min on ice; After ultrasonication, high speed centrifugation 12000rpm, 10min, 4 DEG C;
(5) collect the supernatant that step (4) obtains, joined in nickel ion affinity chromatograph, after being combined with column material, with Washing buffer (50mM NaH
2pO4,300mM NaCl, 150mM imidazole, pH8.0) wash away foreign protein, with Elution buffer (50mM NaH
2pO4,300mM NaCl, 300mM imidazole, pH8.0) collect desired Vx3 (A7) albumen, frozen in-20 DEG C, for subsequent use.
Two, the extracting method of ubiquitin protein:
(1) cultivation of tumour cell: the EL4 cell of recovering frozen, 40 DEG C of water-baths add the DMEM substratum containing 10% (v/v) foetal calf serum, 100U/ml penicillin and 100U/ml Streptomycin sulphate immediately after melting, re-suspended cell to 1 × 10
6individual/ml, and moved in 500ml Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO
2constant incubator in cultivate, within every 1 ~ 2 day, change liquid once, conventional 0.25% tryptic digestion goes down to posterity;
(2) process of tumour cell:
Cultivate by step (1) method, treat Growth of Cells to 80%, add rapamycin, Bortezomib and ammonium chloride, add-on is respectively 120nmol/L, 240nmol/L and 35mmol/L, intervenes tumour cell 18 hours, suppresses the degraded of ubiquitin protein;
(3) ubiquitin protein is extracted:
By step (2) gained cell after cell pyrolysis liquid process, to be centrifugally precipitated, to get supernatant liquor and same Vx3 (A7) albumen is hatched altogether, can ubiquitin protein be obtained through nickel ion affinity chromatograph.
Embodiment 3:(tumour cell is EL4 cell)
One, the extracting method of Vx3 (A7) albumen:
(1) pUbiG101-Vx3 (A7)-eGFP plasmid is proceeded to E. coli (DH5-α);
(2) intestinal bacteria picking mono-clonal step (1) obtained, LB (Luria-Bertani) culture medium culturing also does double digestion qualification;
(3) the bacterium enlarged culturing after step (2) being identified, IPTG (Isopropyl β-D-1-thiogalactopyranoside is added when light absorption value is 0.5 under bacterium liquid 600nm wavelength, isopropyl-β-D-thiogalactoside(IPTG)) induce 16h, high speed centrifugation 8000rpm, 15min, supernatant discarded;
(4) precipitation Bing buffer (the 50mM NaH will obtained after step (3) high speed centrifugation
2pO
4, 300mMNaCl, 25mM imidazole, pH8.0) and resuspended, add N,O-Diacetylmuramidase and react 30min on ice; After ultrasonication, high speed centrifugation 12000rpm, 10min, 4 DEG C;
(5) collect the supernatant that step (4) obtains, joined in nickel ion affinity chromatograph, after being combined with column material, with Washing buffer (50mM NaH
2pO4,300mM NaCl, 150mM imidazole, pH8.0) wash away foreign protein, with Elution buffer (50mM NaH
2pO4,300mM NaCl, 300mM imidazole, pH8.0) collect desired Vx3 (A7) albumen, frozen in-20 DEG C, for subsequent use.
Two, the extracting method of ubiquitin protein:
(1) cultivation of tumour cell: the EL4 cell of recovering frozen, 40 DEG C of water-baths add the DMEM substratum containing 10% (v/v) foetal calf serum, 100U/ml penicillin and 100U/ml Streptomycin sulphate immediately after melting, re-suspended cell to 1 × 10
6individual/ml, and moved in 500ml Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO
2constant incubator in cultivate, within every 1 ~ 2 day, change liquid once, conventional 0.25% tryptic digestion goes down to posterity;
(2) process of tumour cell:
Cultivate by step (1) method, treat Growth of Cells to 80%, add rapamycin, Bortezomib and ammonium chloride, add-on is respectively 100nmol/L, 200nmol/L and 30mmol/L, intervenes tumour cell 16 hours, suppresses the degraded of ubiquitin protein;
(3) ubiquitin protein is extracted:
By step (2) gained cell after cell pyrolysis liquid process, to be centrifugally precipitated, to get supernatant liquor and same Vx3 (A7) albumen is hatched altogether, can ubiquitin protein be obtained through nickel ion affinity chromatograph.
Embodiment 4:(tumour cell is B16-F10 cell)
One, the extracting method of Vx3 (A7) albumen:
(1) pUbiG101-Vx3 (A7)-eGFP plasmid is proceeded to E. coli (DH5-α);
(2) intestinal bacteria picking mono-clonal step (1) obtained, LB (Luria-Bertani) culture medium culturing also does double digestion qualification;
(3) the bacterium enlarged culturing after step (2) being identified, IPTG (Isopropyl β-D-1-thiogalactopyranoside is added when light absorption value is 0.5 under bacterium liquid 600nm wavelength, isopropyl-β-D-thiogalactoside(IPTG)) induce 16h, high speed centrifugation 8000rpm, 15min, supernatant discarded;
(4) precipitation Bing buffer (the 50mM NaH will obtained after step (3) high speed centrifugation
2pO
4, 300mMNaCl, 25mM imidazole, pH8.0) and resuspended, add N,O-Diacetylmuramidase and react 30min on ice; After ultrasonication, high speed centrifugation 12000rpm, 10min, 4 DEG C;
(5) collect the supernatant that step (4) obtains, joined in nickel ion affinity chromatograph, after being combined with column material, with Washing buffer (50mM NaH
2pO4,300mM NaCl, 150mM imidazole, pH8.0) wash away foreign protein, with Elution buffer (50mM NaH
2pO4,300mM NaCl, 300mM imidazole, pH8.0) collect desired Vx3 (A7) albumen, frozen in-20 DEG C, for subsequent use.
Two, the extracting method of ubiquitin protein:
(1) cultivation of tumour cell: the B16-F10 cell of recovering frozen, 40 DEG C of water-baths add the DMEM substratum containing 10% (v/v) foetal calf serum, 100U/ml penicillin and 100U/ml Streptomycin sulphate immediately after melting, re-suspended cell to 1 × 10
6individual/ml, and moved in 500ml Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO
2constant incubator in cultivate, within every 1 ~ 2 day, change liquid once, conventional 0.25% tryptic digestion goes down to posterity;
(2) process of tumour cell:
Cultivate by step (1) method, treat Growth of Cells to 80%, add rapamycin, Bortezomib and ammonium chloride, add-on is respectively 100nmol/L, 200nmol/L and 30mmol/L, intervenes tumour cell 16 hours, suppresses the degraded of ubiquitin protein;
(3) ubiquitin protein is extracted:
By step (2) gained cell after cell pyrolysis liquid process, to be centrifugally precipitated, to get supernatant liquor and same Vx3 (A7) albumen is hatched altogether, can ubiquitin protein be obtained through nickel ion affinity chromatograph.
Embodiment 5 (only add Bortezomib, tumour cell is EL4 cell)
One, the extracting method of Vx3 (A7) albumen:
(1) pUbiG101-Vx3 (A7)-eGFP plasmid is proceeded to E. coli (DH5-α);
(2) intestinal bacteria picking mono-clonal step (1) obtained, LB (Luria-Bertani) culture medium culturing also does double digestion qualification;
(3) the bacterium enlarged culturing after step (2) being identified, IPTG (Isopropyl β-D-1-thiogalactopyranoside is added when light absorption value is 0.5 under bacterium liquid 600nm wavelength, isopropyl-β-D-thiogalactoside(IPTG)) induce 16h, high speed centrifugation 8000rpm, 15min, supernatant discarded;
(4) precipitation Bing buffer (the 50mM NaH will obtained after step (3) high speed centrifugation
2pO
4, 300mMNaCl, 25mM imidazole, pH8.0) and resuspended, add N,O-Diacetylmuramidase and react 30min on ice; After ultrasonication, high speed centrifugation 12000rpm, 10min, 4 DEG C;
(5) collect the supernatant that step (4) obtains, joined in nickel ion affinity chromatograph, after being combined with column material, with Washing buffer (50mM NaH
2pO4,300mM NaCl, 150mM imidazole, pH8.0) wash away foreign protein, with Elution buffer (50mM NaH
2pO4,300mM NaCl, 300mM imidazole, pH8.0) collect desired Vx3 (A7) albumen, frozen in-20 DEG C, for subsequent use.
Two, the extracting method of ubiquitin protein:
(1) cultivation of tumour cell: the EL4 cell of recovering frozen, 40 DEG C of water-baths add the DMEM substratum containing 10% (v/v) foetal calf serum, 100U/ml penicillin and 100U/ml Streptomycin sulphate immediately after melting, re-suspended cell to 1 × 10
6individual/ml, and moved in 500ml Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO
2constant incubator in cultivate, within every 1 ~ 2 day, change liquid once, conventional 0.25% tryptic digestion goes down to posterity;
(2) process of tumour cell:
Cultivate by step (1) method, treat Growth of Cells to 80%, add Bortezomib, add-on is 200nmol/L, intervenes tumour cell 16 hours, suppresses the degraded of ubiquitin protein;
(3) ubiquitin protein is extracted:
By step (2) gained cell after cell pyrolysis liquid process, to be centrifugally precipitated, to get supernatant liquor and same Vx3 (A7) albumen is hatched altogether, can ubiquitin protein be obtained through nickel ion affinity chromatograph.
Embodiment 6 (only add Bortezomib, tumour cell is B16-F10 cell)
One, the extracting method of Vx3 (A7) albumen:
(1) pUbiG101-Vx3 (A7)-eGFP plasmid is proceeded to E. coli (DH5-α);
(2) intestinal bacteria picking mono-clonal step (1) obtained, LB (Luria-Bertani) culture medium culturing also does double digestion qualification;
(3) the bacterium enlarged culturing after step (2) being identified, IPTG (Isopropyl β-D-1-thiogalactopyranoside is added when light absorption value is 0.5 under bacterium liquid 600nm wavelength, isopropyl-β-D-thiogalactoside(IPTG)) induce 16h, high speed centrifugation 8000rpm, 15min, supernatant discarded;
(4) precipitation Bing buffer (the 50mM NaH will obtained after step (3) high speed centrifugation
2pO
4, 300mMNaCl, 25mM imidazole, pH8.0) and resuspended, add N,O-Diacetylmuramidase and react 30min on ice; After ultrasonication, high speed centrifugation 12000rpm, 10min, 4 DEG C;
(5) collect the supernatant that step (4) obtains, joined in nickel ion affinity chromatograph, after being combined with column material, with Washing buffer (50mM NaH
2pO4,300mM NaCl, 150mM imidazole, pH8.0) wash away foreign protein, with Elution buffer (50mM NaH
2pO4,300mM NaCl, 300mM imidazole, pH8.0) collect desired Vx3 (A7) albumen, frozen in-20 DEG C, for subsequent use.
Two, the extracting method of ubiquitin protein:
(1) cultivation of tumour cell: the B16-F10 cell of recovering frozen, 40 DEG C of water-baths add the DMEM substratum containing 10% (v/v) foetal calf serum, 100U/ml penicillin and 100U/ml Streptomycin sulphate immediately after melting, re-suspended cell to 1 × 10
6individual/ml, and moved in 500ml Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO
2constant incubator in cultivate, within every 1 ~ 2 day, change liquid once, conventional 0.25% tryptic digestion goes down to posterity;
(2) process of tumour cell:
Cultivate by step (1) method, treat Growth of Cells to 80%, add Bortezomib, add-on is 200nmol/L, intervenes tumour cell 16 hours, suppresses the degraded of ubiquitin protein;
(3) ubiquitin protein is extracted:
By step (2) gained cell after cell pyrolysis liquid process, to be centrifugally precipitated, to get supernatant liquor and same Vx3 (A7) albumen is hatched altogether, can ubiquitin protein be obtained through nickel ion affinity chromatograph.
Embodiment 7 Western blot detects ubiquitin protein
Respectively embodiment 3 and embodiment 4 gained ubiquitin protein are added SDS-PAGE albumen sample-loading buffer (the green skies, Jiangsu, P0015), 15% polyacrylamide gel electrophoresis is separated, and adds against ubiquitin antibody (anti-ub antibody), adds two anti-colour developings.
1, preparative separation glue and concentrated glue: fill a prescription as follows
Prepared by A separation gel (15%):
B concentrates the preparation of glue (5%):
2, SDS-PAGE (polyacrylamide gel electrophoresis)
After gelling to be concentrated is solid, comb hole with deionized water rinsing.According to protein quantification result adjustment applied sample amount, the every hole of cell lysate is about 12ml, and add 5 × SDS sample loading buffer 3ml and mix, 100 DEG C are boiled 5min, abandons sediment and get supernatant preparation loading after the centrifugal 5min of 12000rpm.Add Protein Marker (the green skies, Jiangsu, P0069), in upper and lower groove, add Tris-glycine running buffer, start electrophoresis, constant voltage 80V, enters after separation gel until sample, high voltage, to 120V, stops electrophoresis when tetrabromophenol sulfonphthalein moves to bottom offset plate.
3, transferring film
Transferring film starts precontract half an hour, transfer device is soaked in Western transferring film liquid (the green skies, Jiangsu of precooling, P0021A) in, after electrophoresis terminates, offset plate is taken off, the glue of corresponding size is cut according to target protein molecular weight, be soaked in transfering buffering liquid, cut PVDF (polyvinylidene difluoride (PVDF)) film of suitable size according to the size of glue, be soaked in methanol solution, move to after film soaks in transfering buffering liquid and continue to soak 20min, preparation " sandwich ".Constant current 250mA, electrotransfer 60min.
4, close
After 0.05%TBST (the green skies, Jiangsu, P0023C) washing by soaking pvdf membrane 5min, be placed in and shake closed 1 hour slowly containing 3% bovine serum albumin Western washings room temperature
5, primary antibodie is hatched
TBST1:1000 dilutes anti-Ub rabbit against murine monoclonal antibody, and 4 DEG C are spent the night.Next day, TBST washed 3 times, each washing 10 minutes, cradle vibrate amplitude 120 revs/min
6, two anti-hatch and develop
TBST 1:5000 dilutes the goat anti-rabbit igg antibody that HRP (horseradish peroxidase) marks, room temperature, and shaking table hatches 1 hour; TBST washs 3 times, each washing 10 minutes, cradle vibrate amplitude 120rpm; With the super quick luminous substrate exposure imaging of ECL.
As shown in Figure 1, from the lysate of EL4 cell (A) and B16-F10 (B) cell, a large amount of ubiquitin proteins has all been extracted.
Embodiment 8 ubiquitin protein inducing tumor specific immune reacts
(1) 12 C57BL/6J mouse are divided into following 4 groups at random, often organize 3: the amount of its injection embodiment 3 gained ubiquitin protein is respectively 0ug/ mouse, 30ug/ mouse, 100ug/ mouse, 300ug/ mouse;
(2) carried out subcutaneous injection respectively at the 1st, 5,9 day, each group injects the albumen that 1ml total protein content is 0 μ g/ml, 30 μ g/ml, 100 μ g/ml, 300 μ g/ml respectively;
(3) the 12nd days, mouse cervical dislocation is put to death, in 75% ethanol, soaks 5min, cut with aseptic operation and tweezers taking-up mouse spleen, filter through 200 order metal screen grindings, prepare single cell suspension, count and use the suspension of respective amount substratum to make its cell density be 2.5 × 10
6individual/ml.
(4) respectively get lymphocyte 1ml to 24 orifice plate that these 4 kinds of different modes immune group obtain, add the EL7 cell of 0.5 × 106 deactivation or anti-CD49d McAb (positive control exists) or RPMI (Roswell ParkMemorial Institute) RPMI-1640 (negative control) respectively again and carry out stimulated in vitro;
(5) cultivate after 72 hours, collect supernatant, ELISA (Aenzyme linked immunosorbent assay) detects the amount of secretion of gamma-IFN in supernatant (Interferon-γ).
As shown in Figures 2 and 3, can produce IFN-γ (IFN-γ react the cytokine produced for tumor-specific immunity) with the EL4 cytositimulation Mouse spleen cells of deactivation after the ubiquitin protein of injection EL4 cell derived, and the amount that its amount produced injects together ubiquitin protein is directly proportional.
The tumor-specific immunity reaction of the different immunization ways induction of embodiment 9
(1) 6 C57BL/6J mouse are divided into following 2 groups at random, one group adopts lymphoglandula immunity (1 time) to add subcutaneous inoculation (3 times), and one group adopts three subcutaneous inoculations;
(2) carried out immunity respectively at the 1st, 5,9 day, each group injects the embodiment 3 gained ubiquitin protein that 1ml total protein content is 100 μ g/ml respectively;
(3) the 12nd days, mouse cervical dislocation is put to death, in 75% ethanol, soaks 5min, cut with aseptic operation and tweezers taking-up mouse spleen, filter through 200 order metal screen grindings, prepare single cell suspension, count and use the suspension of respective amount substratum to make its cell density be 2.5 × 10
6individual/ml.
(4) respectively get lymphocyte 1ml to 24 orifice plate that these 4 kinds of different modes immune group obtain, add 0.5 × 10 more respectively
6the E.G7 cell of deactivation carries out stimulated in vitro;
(5) cultivate after 72 hours, collect supernatant, ELISA detects the amount of secretion of gamma-IFN in supernatant.
As shown in Figure 4, lymphoglandula immunity (1 time) adds subcutaneous inoculation (2 times) and compares with employing 3 subcutaneous inoculations, can produce more IFN-γ with after the EL4 cytositimulation Mouse spleen cells of deactivation.
The knurl specific immune response that embodiment 10 ubiquitin protein and DRibble induce
(1) 9 C57BL/6J mouse are divided into following 3 groups at random, one group is PBS group (control group), and one group is ubiquitin protein matter group, and one group is Dribble group;
(2) carried out immunity respectively at the 1st, 5,9 day, each group injects ubiquitin protein or the Dribble that 1ml total protein content is 100 μ g/ml respectively, control group injection PBS;
(3) the 12nd days, mouse cervical dislocation is put to death, in 75% ethanol, soaks 5min, cut with aseptic operation and tweezers taking-up mouse spleen, filter through 200 order metal screen grindings, prepare single cell suspension, count and use the suspension of respective amount substratum to make its cell density be 2.5 × 10
6individual/ml.
(4) respectively get lymphocyte 1ml to 24 orifice plate that these 4 kinds of different modes immune group obtain, add 0.5 × 10 more respectively
6the E.G7 cell of deactivation carries out stimulated in vitro;
(5) cultivate after 72 hours, collect supernatant, ELISA detects the amount of secretion of gamma-IFN in supernatant.
As depicted in figures 5 and 6, after ubiquitin protein (being respectively embodiment 3 and embodiment 4 gained) and Dribble immune mouse, IFN-γ can be produced with after the EL4 cytositimulation Mouse spleen cells of deactivation.
The knurl specific immune response intercrossing (using embodiment 5 and embodiment 6 gained ubiquitin protein respectively) of embodiment 11 ubiquitin protein induction
(1) 12 C57BL/6J mouse are divided into following 4 groups at random, one group is PBS group (control group), one group be full cell pyrolysis liquid group, one group be ubiquitin protein matter group, one group is ubiquitin protein rejecting group;
(2) carried out immunity respectively at the 1st, 5,9 day, each group injects the albumen that 1ml total protein content is 100 μ g/ml respectively, control group injection PBS;
(3) the 12nd days, mouse cervical dislocation is put to death, 5min is soaked in 75% ethanol, cut with aseptic operation and tweezers taking-up mouse spleen, filter through 200 order metal screen grindings, prepare single cell suspension, count and use the suspension of respective amount substratum to make its cell density be 2.5 × 106/ml.
(4) respectively get lymphocyte 1ml to 24 orifice plate that these 4 kinds of different modes immune group obtain, add the EL4 cell of 0.5 × 106 deactivation or the B16-F10 of deactivation or anti-cd 3 antibodies respectively again and carry out stimulated in vitro;
(5) cultivate after 72 hours, collect supernatant, ELISA detects the amount of secretion of gamma-IFN in supernatant.
As depicted in figures 7 and 8, and with not containing compared with after ubiquitin protein immune mouse, after ubiquitin protein immune mouse, then use as killed cells process, the EL4 cell of deactivation or the B16-F10 cytositimulation Mouse spleen cells of deactivation can produce more IFN-γ.Further, after the ubiquitin protein immune mouse in EL4 source, IFN-γ can be produced equally with the B16-F10 cytositimulation Mouse spleen cells of deactivation.Therefore, show that ubiquitin protein that tumour is originated can play the effect of cross immunity, namely the ubiquitin protein of EL4 cell derived can cause the immunne response after B16 cytositimulation, and in like manner the ubiquitin protein of B16 cell derived can cause the immunne response after EL4 cytositimulation.But the immune effect of heterologous protein is starkly lower than homologous protein.
The knurl specific immune response intercrossing (using embodiment 3 and embodiment 4 gained ubiquitin protein respectively) of embodiment 12 ubiquitin protein induction
(1) 12 C57BL/6J mouse are divided into following 4 groups at random, one group is PBS group (control group), one group be full cell pyrolysis liquid group, one group be ubiquitin protein matter group, one group is ubiquitin protein rejecting group;
(2) carried out immunity respectively at the 1st, 5,9 day, each group injects the albumen that 1ml total protein content is 100 μ g/ml respectively, control group injection PBS;
(3) the 12nd days, mouse cervical dislocation is put to death, 5min is soaked in 75% ethanol, cut with aseptic operation and tweezers taking-up mouse spleen, filter through 200 order metal screen grindings, prepare single cell suspension, count and use the suspension of respective amount substratum to make its cell density be 2.5 × 106/ml.
(4) respectively get lymphocyte 1ml to 24 orifice plate that these 4 kinds of different modes immune group obtain, add the EL4 cell of 0.5 × 106 deactivation or the B16-F10 of deactivation or anti-cd 3 antibodies respectively again and carry out stimulated in vitro;
(5) cultivate after 72 hours, collect supernatant, ELISA detects the amount of secretion of gamma-IFN in supernatant.
As shown in accompanying drawing 9 and 10, come to the same thing with embodiment 11, the ubiquitin protein of EL4 cell derived can cause the immunne response after B16 cytositimulation, and in like manner the ubiquitin protein of B16 cell derived can cause the immunne response after EL4 cytositimulation; Unlike, embodiment 3 and embodiment 4 method gained ubiquitin protein, the immune effect of heterologous protein is substantially suitable with the immune effect of homologous protein.
Embodiment 13: ubiquitin protein suppresses mouse interior tumor growth
(1) by the subcutaneous injection 1 × 10 respectively of 12 C57BL/6J mouse
6after EL4 cell, be divided into following 4 groups at random, one group is PBS group (control group), one group be full cell pyrolysis liquid group, one group be embodiment 3 gained ubiquitin protein matter group, one group is the ubiquitin protein rejecting group of EL4 cell derived;
(2) carried out immunity respectively at the 1st, 5,9 day, each group injects the albumen that 1ml total protein content is 100 μ g/ml respectively, control group injection PBS;
(3) be the 0th day from injection tumour cell, measured each group of tumor size every 7 days, and observe the survival state of mouse every day;
(4) according to Plotting data tumor size curve and the mouse survival curve of gained;
Result as shown figs. 11 and 12, compare other three groups and obviously diminish, and the survival time is considerably beyond other three groups by its gross tumor volume of the mouse of ubiquitin protein immunity.
Embodiment 14 ubiquitin protein suppresses mouse interior tumor growth to have intercrossing (using embodiment 5 and embodiment 6 gained ubiquitin protein respectively)
One, to the immunization method of EL4 tumor model
(1) by the subcutaneous injection 1 × 10 respectively of 9 C57BL/6J mouse
6after EL4 cell, be divided into following 3 groups at random, one group is PBS group (control group), and one group is EL4 cellular ubiquitination protein groups, and one group is B16-F10 cellular ubiquitination protein groups;
(2) carried out immunity respectively at the 1st, 5,9 day, each group injects the albumen that 1ml total protein content is 100 μ g/ml respectively, control group injection PBS;
(3) be the 0th day from injection tumour cell, measured each group of tumor size every 7 days, and observe the survival state of mouse every day;
(4) according to Plotting data tumor size curve and the mouse survival curve of gained;
Two, to the immunization method of B16-F10 tumor model
(1) by the subcutaneous injection 1 × 10 respectively of 9 C57BL/6J mouse
6after B16-F10 cell, be divided into following 3 groups at random, one group is PBS group (control group), and one group is EL4 cellular ubiquitination protein group, and one group is B16-F10 cellular ubiquitination protein groups;
(2) carried out immunity respectively at the 1st, 5,9 day, each group injects the albumen that 1ml total protein content is 100 μ g/ml respectively, control group injection PBS;
(3) be the 0th day from injection tumour cell, measured each group of tumor size every 7 days, and observe the survival state of mouse every day;
(4) according to Plotting data tumor size curve and the mouse survival curve of gained;
As shown figs. 13 and 14, result shows, and in EL4 tumor model, compares PBS group all obviously diminish with its gross tumor volume of mouse of different ubiquitin protein immunity, and the survival time is considerably beyond PBS group, but the antitumous effect that heterologous protein immunity causes is starkly lower than homologous protein.
As shown in figs 15 and 16, result shows, and in B16-F10 tumor model, compares PBS group all obviously diminish with its gross tumor volume of mouse of different ubiquitin protein immunity, and the survival time is considerably beyond PBS group, but the antitumous effect that heterologous protein immunity causes also is starkly lower than homologous protein.
Embodiment 15 ubiquitin protein suppresses mouse interior tumor growth to have intercrossing (using embodiment 3 and embodiment 4 gained ubiquitin protein respectively)
One, to the immunization method of EL4 tumor model
(1) by the subcutaneous injection 1 × 10 respectively of 9 C57BL/6J mouse
6after EL4 cell, be divided into following 3 groups at random, one group is PBS group (control group), and one group is EL4 cellular ubiquitination protein groups, and one group is B16-F10 cellular ubiquitination protein groups;
(2) carried out immunity respectively at the 1st, 5,9 day, each group injects the albumen that 1ml total protein content is 100 μ g/ml respectively, control group injection PBS;
(3) be the 0th day from injection tumour cell, measured each group of tumor size every 7 days, and observe the survival state of mouse every day;
(4) according to Plotting data tumor size curve and the mouse survival curve of gained;
Two, to the immunization method of B16-F10 tumor model
(1) by the subcutaneous injection 1 × 10 respectively of 9 C57BL/6J mouse
6after B16-F10 cell, be divided into following 3 groups at random, one group is PBS group (control group), and one group is EL4 cellular ubiquitination protein group, and one group is B16-F10 cellular ubiquitination protein groups;
(2) carried out immunity respectively at the 1st, 5,9 day, each group injects the albumen that 1ml total protein content is 100 μ g/ml respectively, control group injection PBS;
(3) be the 0th day from injection tumour cell, measured each group of tumor size every 7 days, and observe the survival state of mouse every day;
(4) according to Plotting data tumor size curve and the mouse survival curve of gained;
As shown in figs 17 and 18, result shows, in EL4 tumor model, compare PBS group with its gross tumor volume of mouse of different ubiquitin protein immunity all obviously to diminish, and the survival time is considerably beyond PBS group, the antitumous effect caused unlike the immunity of, heterologous protein with embodiment 14 result is substantially suitable with homologous protein.
As shown in accompanying drawing 19 and 20, result shows, in B16-F10 tumor model, compare PBS group with its gross tumor volume of mouse of different ubiquitin protein immunity all obviously to diminish, and the survival time is considerably beyond PBS group, the antitumous effect caused unlike the immunity of, heterologous protein with embodiment 14 result is also substantially suitable with homologous protein.
Embodiment 16
Example 3 and 4 gained ubiquitin protein, conventionally makes ubiquitin protein vaccine with nano-class aluminum adjuvant respectively.
Claims (10)
1. an antitumor ubiquitin protein, is characterized in that, it mainly extracts gained by following steps:
(1) cultivation of tumour cell: frozen tumour cell of recovering, conveniently cultural method is cultivated;
(2) process of tumour cell:
Growth of Cells to 80% is treated by step (1) method culturing cell, add rapamycin, Bortezomib and ammonium chloride, add-on is respectively (80-120) nmol/L, (160-240) nmol/L and (25-35) mmol/L, intervene tumour cell 14-18 hour, suppress the degraded of ubiquitin protein;
(3) ubiquitin protein is extracted:
By step (2) gained cell after cell pyrolysis liquid process, to be centrifugally precipitated, to get supernatant liquor and same Vx3 (A7) albumen is hatched altogether, can ubiquitin protein be obtained through nickel ion affinity chromatograph.
2. antitumor ubiquitin protein according to claim 1, is characterized in that, described tumour cell is multiple myeloma cells or melanoma cell.
3. antitumor ubiquitin protein according to claim 1, is characterized in that, the conventional culture methods in described step (1) is as follows:
Recover frozen tumour cell, 40 DEG C of water-baths add the DMEM substratum containing 10% (v/v) foetal calf serum, 100U/ml penicillin and 100U/ml Streptomycin sulphate immediately after melting, re-suspended cell to 1 × 10
6individual/ml, and moved in Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO
2constant incubator in cultivate, within every 1 ~ 2 day, change liquid once, conventional 0.25% tryptic digestion goes down to posterity.
4. antitumor ubiquitin protein according to claim 1, is characterized in that, the extracting method of described Vx3 (A7) albumen comprises the steps:
(1) pUbiG101-Vx3 (A7)-eGFP plasmid is proceeded to E. coli (DH5-α);
(2) intestinal bacteria picking mono-clonal step (1) obtained, LB culture medium culturing also does double digestion qualification;
(3) the bacterium enlarged culturing after step (2) being identified, adds IPTG and induces 16h, high speed centrifugation 8000rpm, 15min, supernatant discarded when light absorption value is 0.5 under bacterium liquid 600nm wavelength;
(4) by resuspended for the precipitation Bing buffer obtained after step (3) high speed centrifugation, add N,O-Diacetylmuramidase and react 30min on ice; After ultrasonication, high speed centrifugation 12000rpm, 10min, 4 DEG C;
(5) collect the supernatant that step (4) obtains, joined in nickel ion affinity chromatograph, after being combined with column material, foreign protein is washed away with Washing buffer, desired Vx3 (A7) albumen is collected with Elution buffer, frozen in-20 DEG C, for subsequent use.
5. the extracting method of antitumor ubiquitin protein described in any one of claim 1-4, is characterized in that, comprise the following steps:
(1) cultivation of tumour cell: frozen tumour cell of recovering, conveniently cultural method is cultivated;
(2) process of tumour cell:
Growth of Cells to 80% is treated by step (1) method culturing cell, add rapamycin, Bortezomib and ammonium chloride, add-on is respectively (80-120) nmol/L, (160-240) nmol/L and (25-35) mmol/L, intervene tumour cell 14-18 hour, suppress the degraded of ubiquitin protein;
(3) ubiquitin protein is extracted:
By step (2) gained cell after cell pyrolysis liquid process, to be centrifugally precipitated, to get supernatant liquor and same Vx3 (A7) albumen is hatched altogether, can ubiquitin protein be obtained through nickel ion affinity chromatograph.
6. the extracting method of antitumor ubiquitin protein according to claim 5, is characterized in that, described tumour cell is multiple myeloma cells or melanoma cell.
7. the extracting method of antitumor ubiquitin protein according to claim 5, is characterized in that, the conventional culture methods in described step (1) is as follows:
Recover frozen tumour cell, 40 DEG C of water-baths add the DMEM substratum containing 10% (v/v) foetal calf serum, 100U/ml penicillin and 100U/ml Streptomycin sulphate immediately after melting, re-suspended cell to 1 × 10
6individual/ml, and moved in Tissue Culture Flask, at 37 DEG C, 5% (V/V) CO
2constant incubator in cultivate, within every 1 ~ 2 day, change liquid once, conventional 0.25% tryptic digestion goes down to posterity.
8. the extracting method of antitumor ubiquitin protein according to claim 5, is characterized in that, the extracting method of described Vx3 (A7) albumen comprises the steps:
(1) pUbiG101-Vx3 (A7)-eGFP plasmid is proceeded to E. coli (DH5-α);
(2) intestinal bacteria picking mono-clonal step (1) obtained, LB culture medium culturing also does double digestion qualification;
(3) the bacterium enlarged culturing after step (2) being identified, adds IPTG and induces 16h, high speed centrifugation 8000rpm, 15min, supernatant discarded when light absorption value is 0.5 under bacterium liquid 600nm wavelength;
(4) by resuspended for the precipitation Bing buffer obtained after step (3) high speed centrifugation, add N,O-Diacetylmuramidase and react 30min on ice; After ultrasonication, high speed centrifugation 12000rpm, 10min, 4 DEG C;
(5) collect the supernatant that step (4) obtains, joined in nickel ion affinity chromatograph, after being combined with column material, foreign protein is washed away with Washing buffer, desired Vx3 (A7) albumen is collected with Elution buffer, frozen in-20 DEG C, for subsequent use.
9. the application of antitumor ubiquitin protein described in any one of claim 1-4, is characterized in that, described ubiquitin protein is preparing the application in tumor therapeutic vaccine.
10. the application of antitumor ubiquitin protein according to claim 9, is characterized in that, described vaccine is combined by ubiquitin protein and nano-class aluminum adjuvant to make.
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| CN105777873A (en) * | 2016-03-25 | 2016-07-20 | 东南大学 | His-Vx3-eGFP protein-nano aluminum covalent coupling ubiquitinated protein containing carbon-nitrogen bonds as well as extraction method and application thereof |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105777910A (en) * | 2016-03-25 | 2016-07-20 | 东南大学 | His-Vx3-eGFP protein-nano aluminum covalent coupling ubiquitinated protein containing peptide bonds as well as extraction method and application thereof |
| CN105777873A (en) * | 2016-03-25 | 2016-07-20 | 东南大学 | His-Vx3-eGFP protein-nano aluminum covalent coupling ubiquitinated protein containing carbon-nitrogen bonds as well as extraction method and application thereof |
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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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