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Application of human recombination protein PDCD5 in preparing tumor chemotherapy sensitizing medicine

A technology of human recombination and protein, which is applied in the direction of anti-tumor drugs, peptide/protein components, drug combinations, etc., and can solve the problems of limiting chemotherapy and the toxic effects of chemotherapy drugs

Inactive Publication Date: 2008-07-09
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Chemotherapy for tumors is still one of the main treatments at present, but due to the toxicity of chemotherapy drugs, the application of chemotherapy is limited.

Method used

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  • Application of human recombination protein PDCD5 in preparing tumor chemotherapy sensitizing medicine
  • Application of human recombination protein PDCD5 in preparing tumor chemotherapy sensitizing medicine
  • Application of human recombination protein PDCD5 in preparing tumor chemotherapy sensitizing medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, construction PDCD5 Escherichia coli expression plasmid

[0032] According to the cDNA and protein sequences of the PDCD5 coding region (see Chinese patent application: ZL98101869.6, publication number CN1218833A), PDCD5 was directional inserted into the pET30a vector (purchased from Novagen) using the NdeI and HindIII sites to obtain the pET30a-PDCD5 expression vector. After the correctness of the plasmid was verified by sequencing, the BL21 strain was transformed.

Embodiment 2

[0033] Embodiment 2, expression and purification of PDCD5 in Escherichia coli

[0034] After the ET30a-PDCD5 expression vector was transformed into BL21(DE3) Escherichia coli and amplified, 1mMIPTG (isopropyl-β-D-thiogalactoside) (Sigma-Aldrich) induced the expression of human recombinant PDCD5 protein, which was anion-exchanged with DEAE Chromatography (Amersham Pharmacia Biotech) and hydrophobic chromatography (Amersham Pharmacia Biotech) to obtain PDCD5 purified protein. used in the following experiments.

[0035] The results are shown in FIG. 1 , by densitometric scanning, the expression level of the target protein PDCD5 is about 8%. Most of the protein was expressed in the supernatant form. As shown in Figure 2, by exploring and optimizing the separation and purification conditions, after two-step purification by ion exchange and hydrophobic chromatography, human recombinant PDCD5 protein with a purity greater than 95% at the electrophoretic level can be obtained.

Embodiment 3

[0036] Embodiment 3, PDCD5 internalization kinetics

[0037] HEK293 and U937 cells (purchased from ATCC) were cultured in RPMI containing 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, USA), 100 U / ml penicillin, 100 μg / ml streptomycin, 2 mM L-glutamic acid 1640 (Life Technologies, USA). HEK293 cells are an adherent-growing cell expressing caveolin-1, and U937 cells are a suspension-growing cell that does not express caveolin-1.

[0038] Human recombinant PDCD5 protein was labeled with fluorescein isothiocyanate (FITC) in 0.025M carbonate buffer (pH9.0), and then purified by Sephadex G25 to remove free FITC , to obtain PDCD5-FITC protein.

[0039] Flow cytometric analysis of the internalization of FITC-labeled PDCD5 recombinant protein: Culture HEK293 and U937 cells to 70% in 24-well plates, and then incubate with 5 μg / ml recombinant PDCD5-FITC. The incubation time points are shown in the figure (Figure 3) . The cells were washed twice with PBS, treated with trypsi...

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Abstract

The present invention relates to the application of human recombined protein PDCD5 in preparing tumor chemotherapy temoporfin. Human recombined protein PDCD5 is non-virus intracellular transfer protein, which enters into cells through lipid raft / caveolae-dependent phagocytosis; PDCD5 can promote the death of programmed cells; the enter of the exogenous human recombined protein PDCD5 into cells can strengthen the sensitivity of various tumor cells to various chemotherapeutic drugs and promote the death of tumor cells caused by chemotherapeutic drugs.

Description

technical field [0001] The invention relates to the application of human recombinant protein PDCD5 in the preparation of tumor chemotherapy sensitizers. Background technique [0002] PDCD5 (programmed cell death 5), previously known as TFAR19 (TF-1 cell apoptosis-related gene 19, TF-1 cell apoptosis-related gene 19), was detected by the expression difference analysis (cDNA Representational Differences Analysis, cDNA-RDA method) cloned (Liu et al. Biochem. Biophys. Res. Commun. 254, 203-210 (1999)), during the process of inducing apoptosis of TF-1 cells by removing cytokines Genes with increased expression. Studies have shown that when PDCD5 is transiently or stably expressed in cell lines, it can promote apoptosis induced by withdrawal of certain stimulators such as growth factors, and enhance TAJ / TROY-induced paraptotic cell death. Chen et al. (Chen, et al. FEBS Lett. 509, 191-196 (2001).) reported that the translocation of PDCD5 from the cytoplasm to the nucleus is an ea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P35/00
CPCA61K38/1761A61K45/06A61P35/00A61P39/00A61K2300/00
Inventor 王应马大龙宋泉声张颖妹娄雅欣克晓燕史琳王艳芳
Owner PEKING UNIV
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