Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Construction, expression and application of tumor-targeted peptide F3 modified cowpea chlorotic mottle virus-like particles

A chlorotic mottle virus and tumor targeting technology, applied in the field of genetic engineering, can solve the problems of lipophilicity and limited application

Inactive Publication Date: 2018-09-21
ECOLOGY INST SHANDONG ACAD OF SCI
View PDF3 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IR780 iodide is a near-infrared dye with higher and more stable fluorescence intensity than indocyanine green (ICG) for clinical use, and can be used for photothermal therapy PTT by laser irradiation, but its lipophilicity limits its application

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction, expression and application of tumor-targeted peptide F3 modified cowpea chlorotic mottle virus-like particles
  • Construction, expression and application of tumor-targeted peptide F3 modified cowpea chlorotic mottle virus-like particles
  • Construction, expression and application of tumor-targeted peptide F3 modified cowpea chlorotic mottle virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of recombinant expression plasmid pPICZ A-F3-CP (ΔN26)

[0046] The tumor-targeting peptide F3 gene was inserted into the CCMVCP (ΔN26) gene with 1-26 amino acid residues deleted at the amino terminal by PCR method to construct the fusion gene F3-CP (ΔN26). The 1-25 amino acid residues at the amino terminus of CCMV CP are the RNA binding domain, and studies have shown that its knockout or replacement does not affect the assembly of CP dimers into VLPs. First, two sets of DNA oligonucleotides Olig-1 and Olig-C1, Olig-2 and Olig-C2 with complementary sequences were designed and annealed to obtain F3 gene and CP(ΔN26) fragments respectively, with 50 overlapping bases between them Base (Table 1). Subsequently, double asymmetric PCR (DA-PCR) and overlap extension PCR (OE-PCR) were used for two-step gene synthesis with corresponding primers (F3-Up and F3-Dn, CP-Up and CP-Dn). The overlapping bases between them serve as bridges. Finally, the fusion ge...

Embodiment 2F3

[0053] Example 2 Induced expression and purification of F3-CCMV virus-like particles

[0054] After linearizing the recombinant plasmid pPICZ A-F3-CP (ΔN26) with SacI, it was transformed into Pichia pastoris GS115 by electroporation. The transformed Pichia pastoris was spread on a YPD plate (1% yeast extract, 2% peptone, 2% glucose, 2% agar powder) containing 100 μg / mL bleomycin Zeocin, cultured upside down at 30°C for 2-3 days, and screened positive transformants.

[0055] The identified positive transformants were inoculated with buffered glycerol complex medium BMGY and buffered methanol complex medium for methanol induction. Collect yeast cells by centrifugation, add an equal volume of 0.5mm acid-washed glass beads (purchased from Sigma) and 5 times the volume of breaking buffer (pH 5.5, 50mM sodium phosphate, 1mM PMSF, 1mM EDTA, 5% glycerol), and vortex broken. Centrifuge to collect the supernatant, carry out protein gel electrophoresis (SDS-PAGE), detect the expressio...

Embodiment 3F3

[0057] Identification of embodiment 3F3-CCMV virus-like particles

[0058] The morphology of the purified F3-CCMV virus-like particles was observed under a transmission electron microscope (nickel mesh, negatively stained with 2% uranyl acetate). virus particles (such as image 3 shown in A). Dynamic light scattering DLS determines that its hydrodynamic diameter is 18.2 ± 1.4nm, has good monodispersity (see image 3 B). This and the CCMV capsid protein CP in the prior art are tagged with histidine (His 6 ) and elastin-like polypeptide (ELP) modified virus-like particles are similar in size, and should be pseudovirus particles composed of 120 CP subunits (T=2), rather than wild CCMV composed of 180 CP subunits Virus particles (T=3).

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Diameteraaaaaaaaaa
Login to View More

Abstract

The invention provides a construction and application of tumor-targeted peptide F3 modified cowpea chlorotic mottle virus-like particles. The F3-CCMV virus-like particles are constructed by insertingthe tumor-targeted peptide F3 into CCMV capsid protein CP whose amino terminal 1-26 amino acid residues are knocked out. The F3-CCMV virus-like particles serve as targeted drug delivery carriers to encapsulate near-infrared dye IR780 iodide, thereby preparing F3-CCMV-IR780 nanoparticles; and the F3-CCMV virus-like particles is capable of targeting and recognizing tumor cells, killing the tumor cells by a photothermal effect, and exerting a biological immune-targeting therapeutic effect.

Description

Technical field: [0001] The invention relates to the field of genetic engineering, and relates to a modification, expression, preparation method and application of plant virus-like particles, in particular to the construction, expression and application of cowpea chlorotic mottle virus-like particles modified with tumor targeting peptide F3. Background technique: [0002] Virus-like particles (Virus-like particles, VLPs) are empty shell structures without viral nucleic acid, which are similar in shape and structure to natural virus particles, and have strong immunogenicity and biological activity. Since VLPs do not contain viral genetic material and are non-infectious, some have been successfully used as vaccines. Many viral structural proteins have the ability to self-assemble into VLPs, which can be produced by heterologous expression systems of non-original hosts. [0003] The nanoparticle carrier constructed on the basis of VLPs has the advantages of good biocompatibili...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/08C12N15/62C12N15/81A61K47/64A61P35/00
CPCA61K47/64A61P35/00C07K14/005C07K14/47C07K2319/00C12N15/815C12N2770/14023
Inventor 吴远征李纪顺扈进冬魏艳丽陈凯杨合同申铉载
Owner ECOLOGY INST SHANDONG ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com