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Polylysine oligomer modified recombined apoferritin nanometer cage and preparation thereof

A technology of oligolysine and apoferritin, which is applied in the field of recombinant apoferritin nanocages and its preparation, can solve the problems of drug degradation and protein cage structure damage, reduce toxic side effects, increase concentration, and improve treatment effect of effect

Inactive Publication Date: 2017-10-24
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, after transcellular transport of unmodified apoferritin nanocages through TfR1 receptor-mediated endocytosis, they usually reside in lysosomes, where a large number of enzymes can cause The destruction of the protein cage structure leads to the degradation and destruction of the drug contained in it when exposed to the lysosome environment. For drugs that need to function in specific organelles in the cytoplasm or in the nucleus, it is necessary to design a Apoferritin Nano-Drug Delivery System with Enzyme Escape Function

Method used

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  • Polylysine oligomer modified recombined apoferritin nanometer cage and preparation thereof
  • Polylysine oligomer modified recombined apoferritin nanometer cage and preparation thereof
  • Polylysine oligomer modified recombined apoferritin nanometer cage and preparation thereof

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Embodiment 1

[0019] Embodiment 1. Construction of pET-30a(+) / n-Lys-FTH expression engineering bacteria

[0020] According to the FTH gene coding sequence to be fully synthesized as reported in the literature, in order to modify the N-terminal of the FTH subunit with different numbers of Lys, add AAGAAAAAGAAA (4-Lys) and AAGAAAAAGAAAAAGAAAAAGAAA (8- Lys), then add CCATGGCT and GCGGCCGC respectively to the 5' end and 3' end of the modified gene coding sequence to obtain Nco I and Not I restriction enzyme recognition sites, and finally the gene sequence is optimized so that the sequence is in Correct expression can be achieved in the E. coli system. The lengths of the two sequences are 580bp and 592bp respectively, and the nucleotide sequences encoding the 4-Lys-HFn and 8-Lys-HFn protein subunits are respectively shown in SEQ ID NO.1 and SEQ ID NO.2. The synthesized sequence was digested with Nco I and Not I, and the plasmid vector pET-30a(+) was subjected to the same double digestion, and t...

Embodiment 2

[0021] Example 2. Analysis of the soluble expression form of the fusion protein of n-Lys-HFn

[0022] Add positive recombinant bacteria to fresh Kan + - In LB medium, at 37°C, 220r / min, shake overnight to activate. Transfer to 5mL Kan at 1% inoculum size + -LB medium, at 37°C, 220r / min, cultured to the OD of the bacterial solution 600 reach 0.5. Add IPTG with a final concentration of 0.1 mM into the test tube, and induce expression for about 6 hours under the same conditions. Take 1mL of bacterial liquid and centrifuge at 8000×g for 3min at 4°C. After discarding the supernatant, add 200 μL of binding buffer (20 mM Tris, 5 mM imidazole, 0.5 M NaCl, pH7.9) to resuspend, resuspend and centrifuge again under the same conditions, discard the supernatant to collect bacteria, and resuspend in 200 μL of binding buffer, frozen overnight at -20°C. After the bacteria were taken out and melted, the bacteria were ultrasonically broken, and the sample was taken out as the total bacter...

Embodiment 3

[0023] A large amount of expression and collection of bacteria of embodiment 3.n-Lys-HFn target fusion protein

[0024] Transfer the positive recombinant bacteria recovered overnight to 100mL Kan + -LB medium, at 37°C, 220r / min, cultured to the OD of the bacterial solution 600reach 0.5. Add IPTG with a final concentration of 0.1 mM into the test tube, and induce expression for about 6 hours under the same conditions. 4°C, 8000×g, centrifuge for 3 minutes to collect the bacteria, add 30mL of binding buffer to resuspend, resuspend and centrifuge again under the same conditions, discard the supernatant to collect the bacteria, resuspend in 30mL of binding buffer, add the final Lysozyme at a concentration of 100 μg / mL, frozen at -20°C.

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Abstract

The invention discloses a polylysine oligomer modified recombined apoferritin nanometer cage and a preparation thereof. The nanometer cage is a recombined apoferritin cage with the surface modified by polylysine oligomer that is acquired in the manner of self-assembling protein subunits into hollow spherical protein and utilizing a genetic recombination expression technique to modify different amount of lysine at the N terminal of the protein subunits. The protein nanometer cage can realize the depolymerization and recombination of the protein subunits by changing the solution pH so as to realize the drug loading; the biocompatibility and the in vivo stability are high; the nanometer cage can specifically recognize the high-expression transferrin receptor on the tumor cell surface, so as to realize the active targeting; after the recombined apoferritin nanometer cage is introduced into the cells under the endocytosis effect, the lysosome escape is realized through the proton sponge effect under the lysosome acid environment under the effect of polylysine residue, so that the drug in the protein cage can be protected from being degraded in the lysosome; the recombined apoferritin nanometer cage is expected to have an excellent application prospect at the aspects of gene drug delivery, drug organelle targeting delivery, and the like.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a recombinant apoferritin nanocage modified by oligolysine and a preparation method thereof. Background technique [0002] Chemotherapy is a common treatment for malignant tumors, but most anticancer drugs have serious toxic and side effects on the body due to the lack of selectivity. Therefore, the development of targeted delivery systems for anti-tumor drugs has extremely important clinical application value. As a natural protein, apoferritin not only has good biocompatibility and stability, but also specifically binds to transferrin receptor 1 (TfR1) highly expressed on the surface of tumor cells, Under the mediated action of the drug, it can effectively enter the cell, so as to achieve the effect of active targeting. Apoferritin is usually self-assembled by 24 subunits to form a hollow spherical cage structure with a particle size of 12-13nm. Utilizing its ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N1/21C12R1/19
CPCC07K14/79C07K2319/21C07K2319/33
Inventor 吴正红马茁袁世睿
Owner CHINA PHARM UNIV
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