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Fusion protein gold nano-cluster complex Au@(gCTX)NCs and preparation method and application thereof

A technology of fusion protein and gold nanoclusters, applied in the field of genetic engineering, to achieve the effects of excellent fluorescence properties, green process, and high fluorescence stability

Inactive Publication Date: 2016-07-27
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Most of the reported methods for synthesizing gold nanoclusters are protected by small molecules or glutathione ligands. There are few studies on the direct synthesis of proteins, mainly focusing on the synthesis of gold nanoclusters using BSA as a template, which is directly applied to MDA. -In vivo non-specific imaging of mouse models of MB-45 and HeLa tumors, gold nanocluster therapeutic drugs for glioma have not been reported yet

Method used

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  • Fusion protein gold nano-cluster complex Au@(gCTX)NCs and preparation method and application thereof
  • Fusion protein gold nano-cluster complex Au@(gCTX)NCs and preparation method and application thereof
  • Fusion protein gold nano-cluster complex Au@(gCTX)NCs and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Molecular design of gCTX protein sequence

[0077] The primary amino acid sequence of the CTX polypeptide obtained by searching the NCBI database is: MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR. According to the primary amino acid sequence of the CTX polypeptide, based on the consideration of the efficient recombinant expression and purification of the scorpion venom polypeptide, it was fused and expressed with the GST protein tag. On this basis, the applicant molecule designed the tumor-targeting protein GST-CTX ( gCTX), whose sequence is the amino acid sequence shown in SEQ ID NO. 1, and the nucleotide coding sequence is shown in SEQ ID NO. 2. Insert the nucleotide coding sequence into the BamH1 and Xho1 sites of the pGEX-6p-1 vector to obtain recombinant expression Plasmid pGEX-CT.

Embodiment 2

[0078] Example 2: Preparation of genetically engineered bacteria Rosetta (DE3) (pGEX-CTX) and expression and affinity chromatography of recombinant chlorotoxin protein (gCTX)

[0079] The recombinant expression plasmid pGEX-CT was transformed into the prepared E. coli competent Rosetta (DE3) cells (see the second edition of "Molecular Cloning" for the method). The plate is LB / AP+agar plate. Monoclonal clones were selected to obtain genetically engineered strain Rosetta (DE3) (KCT-W1 / pGEX-6p-1). Inoculate the clones (recombinant Escherichia coli Rossetta(DE3) / CTX / pGEX-6p-1) in LB liquid medium containing ampicillin at a ratio of 1:100, and add IPTG (final concentration) when cultured at 37℃ to OD6000.8 0.1mM) to induce the culture, and then incubate the culture at 28°C for 4 hours to express the target gene (see figure 1 ). Concentrate the induced culture 50 times, ultrasonically break the cells (80HZ, 30 seconds / time, until the culture becomes clear), centrifuge at 12000rpm for...

Embodiment 3

[0080] Example 3: Concentration desalting and HPLC separation of recombinant gCTX protein

[0081] The fusion protein gCTX eluted in Example 7 was centrifuged and desalted through a 15ml 10KDa ultrafiltration tube (Millipore, Centricon, USA) at a centrifugal speed of 3500rpm / min and a temperature of 4°C. Then the concentrated and desalted gCTX fusion protein was separated by HPLC (product of Angelon, USA). The HPLC parameter settings are: separation column C18column (EliteHPLC, China, 10×250mm, 5μm), flow rate 5ml / min, liquid phase is CH3CN (10% to 80%) eluent containing 0.1% TFA, UV detection is set at 230nm Place. Manually collect gCTX protein peaks (see figure 2 ). Freeze-dry (-40℃), and use the Bradford method to determine the content. B: Preparation of E. coli DH5a and BL21 (DE3) competent cells.

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Abstract

The invention provides a fusion protein gold nano-cluster complex Au@(gCTX)NCs and a preparation method and an application thereof. The technical scheme provided by the invention comprises that a recombinant chlorotoxin protein GST-CTX(gCTX) having a glioma targeting function is used as a template, and the multifunctional chlorotoxin-gold nano-cluster fluorescence complex is synthesized; at the same time, the invention relates to a preparation method of a protein gold nano-cluster complex, and also relates to the tumor targeting specificity of the chlorotoxin-gold nano-cluster fluorescence complex and use of the chlorotoxin-gold nano-cluster fluorescence complex in tumor diagnosis and treatment.

Description

Technical field [0001] The invention relates to the field of genetic engineering, in particular to a fusion protein gold nanocluster complex Au (gCTX) NCs, and a preparation method and application thereof. Background technique [0002] Glioma (glioma), also referred to as glioma, is a primary intracranial tumor that originates from glial cells. Malignant glioma has a high fatality rate, and the early boundary is not obvious. The existing treatment methods are difficult to be effective. Diagnosis and treatment. [0003] In recent decades, through simple and high-resolution optical and chemical methods, targeted cancer diagnosis and treatment technologies have been rapidly developed. Many researchers have focused on optical imaging of cells based on fluorescent nanomaterials. Because of the unique photoelectric and physicochemical properties of fluorescent nanomaterials, they have many advantages over organic dyes and fluorescent proteins, such as autofluorescence intensity, high mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12N1/21G01N21/64C12R1/19
Inventor 蔡林涛孙正博张雯璐龚萍
Owner SHENZHEN INST OF ADVANCED TECH
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