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Method for preparing metal nanoparticle using metal binding protein

A nanoparticle and protein-binding technology, applied in the direction of metallothionein, microbial-based methods, botany equipment and methods, etc., can solve the problem of low penetration of optical imaging

Inactive Publication Date: 2015-11-11
KOREA ADVANCED INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] In addition, in order to overcome the low penetration of optical imaging in vivo, quantum dots with near-infrared wavelengths were used for optical imaging of sentinel lymph nodes in the axilla of rats (Kim et al., Nat. Biotechnol., 22:93, 2004 )

Method used

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  • Method for preparing metal nanoparticle using metal binding protein
  • Method for preparing metal nanoparticle using metal binding protein
  • Method for preparing metal nanoparticle using metal binding protein

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1: Preparation of heavy metal binding protein expression vector

[0052] (1) Preparation of plant chelating peptide synthase expression vector

[0053] In order to obtain the AtPCS gene of synthetic plant chelating peptide synthetase (NCBI accession number AF461180), the Arabidopsis complementary DNA (cDNA) represented by the sequence of base SEQ ID NO: 5 (Arabidopsis plant chelating peptide synthetase) was used As a template, PCR was performed using the primers of SEQ ID NO: 1 and SEQ ID NO: 2. The PCR reaction was performed under the following conditions: pre-denaturation at 94°C for 7 min; denaturation at 94°C for 1 min and 30 cycles; annealing at 55°C for 1 min; extension at 72°C for 1 min; and finally at 72°C for 7 min.

[0054] SEQIDNO:1: 5'-GGAATTCATGGCTATGGCGAGTTTAT-3'

[0055] SEQIDNO:2: 5'-CCCAAGCTTATTAATAGGCAGGAGCAGCGAG-3'

[0056] The 1.5-kb DNA fragment obtained by PCR reaction was separated by agarose gel electrophoresis, and digested with two restriction...

Embodiment 2

[0063] Example 2: Fluorescence analysis of E. coli expressing recombinant plasmid pTJ1-AtPCS

[0064] The plant chelating peptide synthetase was prepared by culturing E.coliDH5α with the transformed recombinant plasmid pTJ1-AtPCSd with a strong inducible tac promoter. For this purpose, the transformed E. coli was inoculated in a 500 mL shake flask containing 100 mL LB liquid medium, which contained heavy metal ions (5mMCdCl 2 ·2.5H 2 O, ZnCl 2 , SeO 2 , TeCL 4 And CsCl 2 ) And incubate at 37°C. The pTJ1-AtPCS recombinant plasmid has a tac promoter, and IPTG is added to induce gene expression. To this end, the culture medium was detected with a spectrophotometer at a wavelength of 600 nm, and when its optical density reached 0.6, 1 mM IPTG was added to induce gene expression. After 4 hours of induction of expression, the medium was centrifuged at 4°C and 6000 rpm for 5 min. Then, the supernatant was discarded, the pellet was washed once with 10 mL PBS buffer (pH 7.4), centrifug...

Embodiment 3

[0066] Example 3: In vivo heavy gold synthesized in E. coli expressing recombinant plasmid pTJ1-AtPCS Genus characteristics

[0067] In order to confirm whether the plant chelating peptide synthetase expressed by IPTG can produce heavy metal structures in E.coliDH5α transformed with the recombinant plasmid pTJ1-AtPCS with inducible tac promoter, the E.coli in Example 2 was washed with distilled water It was dried in a freeze dryer under vacuum for one day, and the heavy metals accumulated in the cells were analyzed by TEM (TehnaiG2, FEI, Netherlands). As a result, it was found that the size and shape of the heavy metal structure were consistent ( Image 6 ). In addition, it can be observed that the heavy metal structure that does not appear in the control group (a) that does not express the plant chelating peptide synthase appears in the E. coli expressing the plant chelating peptide synthetase (b) ( Image 6 b, 6c and 6d).

[0068] The scale bars in the figure can be used to de...

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Abstract

The present invention relates to a method of preparing heavy metal nanoparticles using a heavy metal-binding protein. More specifically, relates to a method for preparing heavy metal structures, comprising the steps of: culturing a microorganism transformed with a gene encoding a heavy metal-binding protein, in a heavy metal ion-containing medium, to produce heavy metal structures in the microorganism; and collecting the produced heavy metal structures, as well as nanoparticles of heavy metal structures prepared according to said method. Unlike prior methods of preparing quantum dots by physically binding metal materials, the quantum dots disclosed herein can be efficiently produced by expressing the heavy metal-binding protein in cells. In addition, the quantum dots are useful because they can solve an optical stability problem that is the shortcoming of organic fluorophores.

Description

[0001] This application is a divisional application of the Chinese invention patent application 200780019089.4 filed on April 17, 2007. Technical field [0002] The present invention relates to a method for preparing heavy metal nanoparticles by using heavy metal binding protein, and in particular to a method for preparing heavy metal structures. The method includes performing heavy metal ion-containing medium on microorganisms transformed with heavy metal binding protein encoding genes. Culturing, thereby producing heavy metal structures in the microorganisms, and collecting the produced heavy metal structures and the nanoparticles of the heavy metal structures produced according to the method. Background technique [0003] Quantum dots are nanometer-scale semiconductor particles that emit light when they are excited by energy such as light, and the color of the light emitted depends on the size of the particles. In other words, if the particle size is reduced to reduce the parti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P3/00C12N15/70C12N15/54C12N15/31C12N1/21G01N33/531G01N33/58C12R1/19
CPCC07K14/825B82Y5/00B82Y15/00C12N9/104C12P3/00G01N33/531G01N33/588Y10T428/2982C12N15/70C12N15/52
Inventor 李相烨朴泰正
Owner KOREA ADVANCED INST OF SCI & TECH
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