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Platinum complexes, protein complexes prepared from same, and preparation method of protein complexes

A technology of complexes and complexes, applied in chemical instruments and methods, pharmaceutical formulations, compounds containing elements of group 8/9/10/18 of the periodic table, etc., to achieve good tumor cell growth inhibitory effect

Inactive Publication Date: 2014-02-05
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been found that it can inhibit the growth of various tumor cells (MCF-7, J774, U2OS cells, etc.), but has no similar effect on the corresponding normal cells

Method used

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  • Platinum complexes, protein complexes prepared from same, and preparation method of protein complexes
  • Platinum complexes, protein complexes prepared from same, and preparation method of protein complexes
  • Platinum complexes, protein complexes prepared from same, and preparation method of protein complexes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: construct the expression vector of azurin

[0038] Use SspI enzyme (purchased from Takara Company) to digest and contain an N-terminal containing (His) 6 -tag, the prokaryotic expression vector pST-GB1 of the GB1 tag containing the TEV restriction site at the C-terminus (see the attached map for the map) figure 1 ), enzyme digestion for 4 hours (see Table 1-1 below for specific conditions). Agarose gel electrophoresis, and gel recovery to obtain the pST-GB1 vector after SspI digestion. Among them, the pST-GB1 vector is a plasmid transformed by our laboratory on the basis of the commercial pET21a. The sequence between the NdeI and BamHI restriction sites on pET21a is excised, and a link containing (His) 6 - a tag, a GB1 solubilizing tag and a sequence of a LIC sequence containing a SspI enzyme cleavage site. With SEQ ID NO: 3 as the forward primer, and SEQ ID NO: 4 as the reverse primer (SEQ ID NO: 3 and SEQ ID NO: 4 were purchased from Shanghai Sangon B...

Embodiment 2

[0050] Example 2: Construction of a molecular cloning vector for an azurin mutant protein by site-directed mutagenesis.

[0051] Using the site-directed mutagenesis technique to the plasmid obtained in Example 1, the histidine on the copper binding site of azurin is mutated into the hydrophobic amino acid glycine to obtain the plasmid of the azurin mutant, and the specific steps are as follows:

[0052] With SEQ ID NO: 5 as the forward primer, and SEQ ID NO: 6 as the reverse primer (SEQ ID NO: 5 and SEQ ID NO: 6 were both purchased from Shanghai Sangon Bioengineering Company), obtained in Example 1 The plasmid was used as a template, using Takara PrimerSTAR TM HS DNA Polymerase with GC Buffer kit for PCR amplification (see Table 2-1 and Table 2-2 for specific conditions); gel recovery of PCR products after agarose gel electrophoresis; process PCR with DpnI enzyme (purchased from Takara) at 37°C The product was produced for 1 hour (see Table 2-3); the digested PCR product was...

Embodiment 3

[0060] Example 3: Expression and purification of azurin mutants

[0061] Transfer the plasmid obtained in Example 2 into BL21 (DE3) competent bacteria, spread the plate, pick a single clone transformant, expand and cultivate to 1 liter of LB medium (each liter of medium contains 0.1 mg of ampicillin) ) cultured; to OD 600 When =0.8, add copper chloride (final concentration is 20 μmol / L) and isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration is 0.2mmol / L), and induce at 25°C for 10 hours .

[0062] Harvest bacteria at 25°C at 4,000 rpm for 20 minutes; resuspend bacteria in Ni-NTA resuspension buffer (30 ml buffer per liter of bacteria); sonicate bacteria at 16,000 rpm at 4°C Centrifuge the cell disruption solution for 30 minutes, and filter the supernatant to obtain the supernatant of the cell disruption solution.

[0063] Pour the supernatant into a Ni-NTA affinity chromatography resin column (Shanghai Sangon Bioengineering Co., Ltd.), and shake on a rotary shak...

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Abstract

The invention discloses tetravalent platinum complexes containing an imidazole group, protein complexes formed by binding the tetravalent platinum complexes and azurin mutant, and a preparation method of the protein complexes. Specifically, the invention relates to azurin and tetravalent platinum complexes, wherein the azurin has a mutant histidine at a copper binding site, and each platinum complex contains an imidazole group, and a protein complex is constructed by coordination of the azurin and the platinum complex. The coordination manner of each tetravalent platinum complex and the azurin mutant is that: the imidazole group of the tetravalent platinum complex is coordinated with the copper atom by replacing the imidazole group of the mutant histidine of the azurin protein, so that the tetravalent platinum complex is coordinated to the copper atom center of the azurin protein mutant. Results of tumor cell growth inhibition experiments show that: the azurin protein contains the tetravalent platinum complex has relatively good tumor-cell growth inhibition effect, and has antitumor drug application prospect.

Description

technical field [0001] The present invention belongs to the field of protein pharmaceutical preparations, and specifically relates to protein pharmaceuticals and a preparation method thereof, in particular to azurin and a class of tetravalent platinum complexes. The azurin has a histidine on its copper binding site To be mutated, the platinum complex used contains an imidazole group, and the two are constructed into a protein complex through coordination binding. Background technique [0002] Cancer has always been synonymous with incurable disease. Since the 21st century, with the development of industrialization, the living environment of human beings has been deteriorating, and the number of cancer patients has also increased year by year. According to data released by the International Union Against Cancer at the 21st World Anti-Cancer Congress in 2010, in 2008, 12.7 million people worldwide suffered from cancer, and the death toll was as high as 7.6 million, accounting...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07F15/00A61K47/48A61K31/555A61P35/00
Inventor 王震刘扬中师红东程芹芹张文星
Owner UNIV OF SCI & TECH OF CHINA
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