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Preparation and application of mitochondrial-targeting self-assembled protein nanoparticles

A nanoparticle and protein technology, applied in peptide/protein components, preparations for in vivo tests, metallothionein, etc., can solve the problems of short half-life, limited application, and ineffective enrichment of small molecules, and achieve biocompatibility Good sex and water solubility, reduced damage, fast targeting effect

Active Publication Date: 2019-03-29
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current drugs that can target mitochondria are mainly small molecules such as triphenylphosphine analogs and mitochondrial membrane-penetrating peptides. However, these small molecules have problems such as short half-lives and ineffective enrichment around tumors. Its application in cancer treatment

Method used

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  • Preparation and application of mitochondrial-targeting self-assembled protein nanoparticles
  • Preparation and application of mitochondrial-targeting self-assembled protein nanoparticles
  • Preparation and application of mitochondrial-targeting self-assembled protein nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, construct the protein nanoparticle amino acid sequence of GSTP1-MT3 mitochondria targeting:

[0056] Based on previous experimental studies, the following mitochondrial-targeted protein nanoparticle expression vectors were constructed, such as figure 1 As shown, GSTP1-MT3 is mainly composed of GSTP1 and MT3, wherein MT3 is at the N-terminus of the amino acid sequence, GSTP1 is at the C-terminus of the amino acid sequence, and MT3 and GSTP1 are coupled through the GGGGS sequence.

[0057] GSTP1 amino acid sequence:

[0058] MPPYTVVYFPVRGRCAALRMLLADQGQSWKEEVVTVETWQEGSLKASCLYGQLPKFQ DGDLTLYQSNTILRHLGRTLGLYGKDQQEAALVDMVNDGVEDLRCKYISLIYTNYEAGKDDYVK ALPGQLKPFETLLSQNQGGKTFIVGDQISFADYNLLDLLLIHEVLAPGCLDAFPLLSAYVGRLSAR PKLKAFLASPEYVNLPINGNGKQ(SEQ IDNO:1);

[0059] MT3 amino acid sequence:

[0060] MDPETCPCPSGGSCTCADSCKCEGCKCTSCKKSCCSCCPAECEKCAKDCVCKGGEAAEAEAEKCSCCQ (SEQ ID NO: 2);

[0061] Linker between GSTP1 and MT3:

[0062] GGGGS (SEQ ID NO: 3).

[0063] Th...

Embodiment 2

[0064] Example 2. Construction of GSTP1-MT3 protein nanoparticle carrier

[0065] In order to facilitate the subsequent expression of GSTP1-MT3 protein nanoparticles in recombinant cells, its prokaryotic expression vector was constructed, wherein pET-28a(+) was selected as the prokaryotic expression vector, and the restriction sites HindIII and NdeI on it were used to convert The nucleotide sequence encoding GSTP1-MT3 shown in SEQ ID NO: 5 was ligated into pET-28a(+), and the recombinant expression vector pET-28a(+)-GSTP1 was successfully obtained after enzyme digestion, electrophoresis and monoclonal sequencing detection -MT3.

Embodiment 3

[0066] Example 3. Recombinant expression of GSTP1-MT3 protein nanoparticles

[0067] Using Escherichia coli as the host bacteria for recombinant expression, the specific expression method is as follows:

[0068] 1. Plasmid transformation

[0069] Take 2 μL 42ng / μL pET-28a(+)-GSTP1-MT3 plasmid, add it to 20 μL BL21(DE3) competent cells, pre-mix on ice for 15-30 minutes, then place in a 42°C water bath and heat for 90 seconds. Then keep on ice for another 10 minutes. Add 800 μL of non-resistant LB medium, incubate at 37°C 220 rpm for 1 hour, then centrifuge at 3500 rpm for 10 minutes, remove 600 μL of supernatant, and mix the remaining 200 μL for use.

[0070] 2. Resistance screening

[0071] Add the remaining 200 μL of the bacterial solution in step 1 to the agarose plate containing kanamycin, incubate in a 37°C incubator for 2 hours, and incubate the plate upside down overnight.

[0072] 3. Monoclonal selection

[0073] A single clone was selected, added to 10 mL of LB me...

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Abstract

The invention relates to mitochondrial-targeting protein nanoparticles, and relates to an amino acid sequence, a coding nucleic acid sequence, a vector of the coding nucleic acid, host expression bacteria and other related information. Under the induction of metal ions, the self-assembled protein nanoparticles can be obtained by an escherichia coli expression system. The protein nanoparticles canbe applied in cancer diagnosis and treatment. Compared with conventional mitochondrial-targeting small molecules (TPP, MPP and the like), the protein nanoparticles can achieve properties of tumor enrichment, mitochondrial targeting, causing of increase of the ROS content in cells, induction of cell apoptosis, inhibition of tumor growth and the like.

Description

technical field [0001] The present invention relates to the fields of bioengineering and medicine, in particular to the technical fields of tumor imaging and treatment and drug delivery, and in particular to a novel mitochondria-targeted protein nanoparticle (GSTP1-MT3). Functional disturbances, enrichment around tumors, and tumor growth inhibition are associated properties. Background technique [0002] Cancer is currently a worldwide problem that people need to solve urgently. According to the statistics of the World Health Organization, nearly 14 million people die of cancer every year, and breast cancer is the second most common cancer in women after skin diseases. According to the US Department of Health statistics, nearly 260,000 women die of breast cancer each year. Therefore, more and more people pay attention to the treatment of breast cancer. [0003] However, patients usually discover their own related symptoms in the late stage of cancer. At the same time, many...

Claims

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Application Information

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IPC IPC(8): A61K47/69A61K47/64A61K47/65A61K38/16A61K31/337A61P35/00A61K49/00A61K51/08A61K51/12A61K49/14A61K49/18
CPCA61K38/16A61K47/64A61K47/65A61K47/6929A61K49/0032A61K49/0056A61K49/0093A61K49/14A61K49/1866A61K51/08A61K51/1244A61P35/00A61K31/337A61K2300/00C12N9/0051C12N9/1088C07K14/00C07K14/825C12N5/0012C12N15/00C12N15/113
Inventor 林坚朱新杰徐良许诺陈龙
Owner PEKING UNIV
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