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
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

IR780 iodide is a near-infrared dye with higher and more stable fluorescence intensity than indocyanine green (ICG) for cl

Method used

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  • 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
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Example Embodiment

[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 whose amino terminal 1-26 amino acid residues were knocked out by PCR method to construct the fusion gene F3-CP (ΔN26). The amino acid residues 1-25 at the amino terminus of CCMV CP are RNA binding domains. Studies have shown that their deletion or substitution does not affect the assembly of CP dimers into VLPs. First, design two sets of DNA oligonucleotides Olig-1 and Olig-C1, Olig-2 and Olig-C2 with complementary sequences, and annealed them to obtain F3 gene and CP(ΔN26) fragments respectively. There are 50 overlapping bases between the two. Base (Table 1). Subsequently, the corresponding primers (F3-Up and F3-Dn, CP-Up and CP-Dn) were used for two-step gene synthesis of double asymmetric PCR (DA-PCR) and overlap extension PCR (OE-PCR). The overlapping bases between the two are used as bridges. Finally, t...

Example Embodiment

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

[0054] After the recombinant plasmid pPICZ A-F3-CP (ΔN26) was linearized with SacI single restriction enzyme digestion, electroporation was used to transform Pichia pastoris GS115. The transformed Pichia pastoris was coated on YPD plates (1% yeast extract, 2% peptone, 2% glucose, 2% agar powder) containing 100μg / mL bleomycin Zeocin, incubated inverted at 30°C for 2-3 days, then 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 pickled glass beads (purchased from Sigma) and 5 times volume of disruption buffer (pH 5.5, 50mM sodium phosphate, 1mM PMSF, 1mM EDTA, 5% glycerol), vortex and shake broken. The supernatant was collected by centrifugation and subjected to protein gel electr...

Example Embodiment

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

[0058] Morphological observation of the purified F3-CCMV virus-like particles under TEM (nickel mesh, 2% uranyl acetate negative staining) showed that F3-CCMV is a spherical nanoparticle with a diameter of about 18nm, which is smaller than wild-type CCMV Virus particles (e.g. image 3 Shown in A). The hydrodynamic diameter measured by dynamic light scattering DLS is 18.2±1.4nm, which has good monodispersity (see image 3 B). This is in contrast to the prior art CCMV capsid protein CP being histidine-tagged (His 6 ) Is similar to the size of the virus-like particle obtained after modification with elastin-like polypeptide (ELP), and should be a pseudovirus particle composed of 120 CP subunits (T=2), while non-wild CCMV is composed of 180 CP subunits Virus particles (T=3).

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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

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Application Information

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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
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