Anti-tumor ubiquitinated protein and extraction method and application thereof

A technology of ubiquitinated protein and extraction method, which is applied in the fields of antitumor drugs, chemical instruments and methods, and pharmaceutical formulations, and can solve the asymmetry between ubiquitinated short-lived proteins and long-lived proteins, poor anti-tumor effect of tumor vaccines, Ubiquitinated short-lived protein degrades quickly and other problems, to achieve the effect of inhibiting tumor growth, overcoming asymmetry and improving the effect

Inactive Publication Date: 2015-09-02
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Therefore, the following problems need to be solved in the prior art, such as poor cross-immunity effect, rapid degradation of ubiquitinated short-lived proteins, and diffi...

Method used

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  • Anti-tumor ubiquitinated protein and extraction method and application thereof
  • Anti-tumor ubiquitinated protein and extraction method and application thereof
  • Anti-tumor ubiquitinated protein and extraction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] 1. Extraction method of Vx3(A7) protein:

[0074] (1) Transform the pUbiG101-Vx3(A7)-eGFP plasmid into Escherichia coli E.coli(DH5-α);

[0075] (2) pick a single clone of the Escherichia coli obtained in step (1), culture it in LB (Luria-Bertani) medium, and perform double enzyme digestion identification;

[0076] (3) Expand the culture of the bacteria identified in step (2), and add IPTG (Isopropylβ-D-1-thiogalactopyranoside, isopropyl-β-D-thiogalactopyranoside) when the absorbance value of the bacteria solution is 0.5 ) for 16 hours of induction, high-speed centrifugation at 8000 rpm for 15 minutes, and discarding the supernatant;

[0077] (4) Use Bing buffer (50mM NaH 2 PO 4 , 300mM NaCl, 25mM imidazole, pH8.0) resuspended, added lysozyme to react on ice for 30min;

[0078] (5) collect the supernatant that step (4) obtains, join it in the nickel ion affinity chromatography, after treating to combine with column material, wash buffer (50mM NaH 2 PO4, 300mM NaCl, ...

Embodiment 2

[0086] 1. Extraction method of Vx3(A7) protein:

[0087] (1) Transform the pUbiG101-Vx3(A7)-eGFP plasmid into Escherichia coli E.coli(DH5-α);

[0088] (2) pick a single clone of the Escherichia coli obtained in step (1), culture it in LB (Luria-Bertani) medium, and perform double enzyme digestion identification;

[0089] (3) Expand the culture of the bacteria identified in step (2), and add IPTG (Isopropylβ-D-1-thiogalactopyranoside, isopropyl-β-D-thiogalactopyranoside) when the absorbance value of the bacteria solution is 0.5 ) for 16 hours of induction, high-speed centrifugation at 8000 rpm for 15 minutes, and discarding the supernatant;

[0090] (4) Use Bing buffer (50mM NaH 2 PO 4 , 300mM NaCl, 25mM imidazole, pH8.0) resuspended, added lysozyme to react on ice for 30min;

[0091] (5) collect the supernatant that step (4) obtains, join it in the nickel ion affinity chromatography, after treating to combine with column material, wash buffer (50mM NaH 2 PO4, 300mM NaCl, 15...

Embodiment 3

[0098] Embodiment 3: (tumor cells are EL4 cells)

[0099] 1. Extraction method of Vx3(A7) protein:

[0100] (1) Transform the pUbiG101-Vx3(A7)-eGFP plasmid into Escherichia coli E.coli(DH5-α);

[0101] (2) pick a single clone of the Escherichia coli obtained in step (1), culture it in LB (Luria-Bertani) medium, and perform double enzyme digestion identification;

[0102] (3) Expand the culture of the bacteria identified in step (2), and add IPTG (Isopropylβ-D-1-thiogalactopyranoside, isopropyl-β-D-thiogalactopyranoside) when the absorbance value of the bacteria solution is 0.5 ) for 16 hours of induction, high-speed centrifugation at 8000 rpm for 15 minutes, and discarding the supernatant;

[0103] (4) Use Bing buffer (50mM NaH 2 PO 4 , 300mM NaCl, 25mM imidazole, pH8.0) resuspended, added lysozyme to react on ice for 30min;

[0104] (5) collect the supernatant that step (4) obtains, join it in the nickel ion affinity chromatography, after treating to combine with column ...

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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 [0001] The invention relates to a ubiquitinated protein and its extraction method and application, belonging to the technical field of tumor vaccines. Background technique [0002] Tumors seriously threaten human health. Curative resection, chemotherapy, and radiotherapy are currently the preferred treatment methods for cancer patients. Clinical data show that most cancer patients are not sensitive to chemotherapy, radiotherapy and other treatments. Therefore, the clinical treatment of tumors urgently needs new methods and means. As a new tumor treatment strategy, tumor biotherapy has the advantages of strong specificity and less toxic side effects, and has attracted more and more attention. Among them, tumor vaccines based on dendritic cells (DC) have become a research hotspot in tumor biotherapy. [0003] In the process of tumor development, the ineffective presentation of tumor antigens to T cells is an important reason for immune escape. At the same t...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K1/14A61K39/00A61P35/00
Inventor 王立新
Owner SOUTHEAST UNIV
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