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Compounds for intracellular delivery of molecules

A complex, cargo molecule technology, applied in the field of molecular biology

Pending Publication Date: 2021-01-29
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the endocytic vesicle escape efficiency of the CPPs macromolecule intracellular delivery system fused with pH-sensitive peptides is indeed improved, a considerable part of the transported macromolecules still remains in the endocytic vesicles (with fluorescent proteins as transported Obvious dot-like distribution can be seen for macromolecules)

Method used

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  • Compounds for intracellular delivery of molecules
  • Compounds for intracellular delivery of molecules
  • Compounds for intracellular delivery of molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0171] Example 1: Evaluation of the endocytic vesicle escape efficiency of the delivery system based on the Split-GFP system

[0172] In the Split-GFP system, the 11 β sheets of GFP are split into large fragments (β1-10) and small fragments (β11), both of which lose their fluorescent activity, but can spontaneously associate and restore GFP if they meet fluorescence properties. Based on this, we constructed HEK293T cells stably expressing Histone-β11, and expressed GFPβ1-10 with nuclear import signal (NLS) as Cargo fusion with the cell-entry delivery system to be evaluated, and then transduced the stable cell line. When the delivery system transduces GFPβ1-10, it can only combine with GFPβ11 to generate complete GFP after successfully escaping from endocytic vesicles and entering the cytoplasm or nucleus. Efficiency is evaluated ( figure 1 ).

[0173] 1.1 Construction of delivery system-GFPβ1-10 protein complex expression vector

[0174] Construct TAT (SEQ ID NO:10), INF7...

Embodiment 2

[0195] Example 2: Application of delivery system in transducing zinc finger protein ZFP

[0196] 2.1 Delivery system-construction of zinc finger protein ZFP9 complex expression vector

[0197] A recombinant protein expression vector containing TAT, INF7, protease cleavage site, and cargo molecule ZFP9 (SEQID NO:27) carrying a nuclear localization signal (NLS) was constructed. The structural schematic diagram of each recombinant protein is as follows Figure 12 As shown, the amino acid sequence of each component is shown in Table 4 below. The construction method is as follows: First, the nucleic acid sequences encoding TAT, INF7, protease cleavage site, and cargo molecule ZFP9 in the delivery system are obtained by PCR amplification, and the various parts are connected through multiple rounds of PCR, and in the last round of PCR In the process, an NdeI restriction site and its overlapping sequence upstream of the NdeI restriction site corresponding to pET-21b(+) were introduce...

Embodiment 3

[0207] Example 3: Application of delivery system in transducin phosphatase Ppm1b

[0208] After TNF-α binds to cell surface receptors, it induces RIP3 phosphorylation and forms a multiprotein complex necrosome (Necrosome). Phosphorylated RIP3 in the necrosome recruits and phosphorylates Mlk1, and the cell enters the necrosis program. During this process, intracellular protein phosphatase 1B (Ppm1b) can dephosphorylate RIP3 to inhibit Necroptosis. In view of the fact that programmed cell necrosis has been found to be closely related to the occurrence of inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases and other diseases, Ppm1b protein has shown great potential in the treatment of the above-mentioned diseases related to programmed cell necrosis .

[0209] 3.1 Delivery system-Ppm1b protein complex expression vector construction

[0210] A recombinant protein expression vector containing TAT, INF7, protease cleavage site, and cargo molecule Ppm1b (S...

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Abstract

The invention relates to the field of molecular biology, in particular to a fusion protein and a compound containing the fusion protein. The fusion protein and the compound can be used for intracellular delivery of cargo molecules. The fusion protein and the compound provided by the invention can realize efficient release of cargo molecules from endocytosis vesicles, and significantly improve thecytoplasm delivery efficiency of the cargo molecules. Once the cargo molecules are available in the cytoplasm, cargo molecules can exert any effect associated therewith. Therefore, the fusion proteinand the compound of the invention provide an effective means for affecting the biological mechanisms and pathways of cells, and can be used in many fields such as research, treatment, diagnosis, and the like.

Description

technical field [0001] The present invention relates to the field of molecular biology, and more specifically, relates to fusion proteins and complexes used for intracellular delivery of cargo molecules. Background technique [0002] Cell membranes are critical to cell survival and function as selectively permeable barriers, and although small molecules can cross cell membranes through natural processes of the cell or by direct diffusion of lipid bilayers, in most cases exogenous Efficient passage of intracellular cargo such as active biomacromolecules from the plasma membrane remains a major obstacle in the cellular transport process. Therefore, a molecular transport tool that can effectively improve the transport efficiency of intracellular loads to living cells is extremely important for its application in biomedicine and other fields. [0003] Cell-penetrating peptides (CPPs) are currently one of the most common and effective carriers used to achieve cellular uptake. C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/87A61K47/42A61K47/64A61K45/00A61P1/16A61P9/10A61P25/28
CPCC07K14/00C12N15/87A61K47/42A61K47/64A61K45/00A61P1/16A61P9/10A61P25/28C07K2319/10C07K2319/50C07K2319/70A61K38/00C12N2310/20C12N15/70C12Y304/21075C12Y304/22034C07K2319/01C12N9/22C07K14/4703C12N9/16C12Y301/03016C07K2319/06C07K2319/09C07K2319/60C07K2319/73C12N15/113C12N2740/16043C07K14/005C07K14/47C12N15/62A61K47/50C12N9/641C07K2319/03C12N2760/16121C12N2760/16122A61K38/465C07K2319/81C12N15/85C12N2760/16022
Inventor 葛胜祥于思远杨晗潘海峰任书玲李廷栋郭清顺熊君辉张军夏宁邵
Owner XIAMEN UNIV