Multimerization delivery system for intracellular delivery of molecules
A technology of multimerization and cells, applied in the field of molecular biology, can solve problems such as research reports, achieve the effects of improving endocytic efficiency, improving vesicle release efficiency, and improving delivery efficiency
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Embodiment 1
[0180] Example 1: Establishment and performance evaluation of TL multimerization delivery system
[0181] In this example, leucine zipper was used to establish a multimerization delivery system, and the effect of multimerization on endocytic efficiency was observed by delivering green fluorescent protein eGFP. By delivering GFPβ1-10-NLS (GFPβ1-10 for short) and based on Split - The GFP evaluation method observes the effect of multimerization on the escape efficiency of vesicles, so as to comprehensively evaluate the effect of multimerization on the delivery efficiency of the delivery system. At the same time, by adding serum (FBS) to the system, the difference in endocytic efficiency before and after multimerization was observed, so as to evaluate the change of serum tolerance.
[0182] 1.1 Construction of multimerized delivery system (TAT-Leu Zipper)-cargo molecule complex expression vector
[0183] Construct the expression vector of the recombinant protein that comprises TA...
Embodiment 2
[0210] Example 2: Establishment and performance evaluation of TINNeL multimerization delivery system
[0211] In this example, on the basis of the multimerization delivery system in Example 1, a pH-sensitive peptide (INF7) and endocytic vesicle protease-specific cleavage sites (CTSL protease: N, Furin protease: Ne) were further added to construct TINNEL delivery system, and its endocytic efficiency and vesicle escape efficiency were evaluated.
[0212] 2.1 Construction of TINNEL-cargo molecule complex expression vector
[0213] The expression vector of the TINNEL-cargo molecule recombinant protein was constructed, and the amino acid sequences of each component contained in each recombinant protein from the N-terminal to the C-terminal are shown in the table below. The construction method is as follows: First, the nucleic acid sequences encoding TAT, INF7, leucine zipper, N, Ne, and cargo molecules (eGFP or GFPβ1-10) in the delivery system are obtained by PCR amplification, an...
Embodiment 3
[0224] Example 3: Application of TINNeL-Ppm1b in inhibiting TNF-α-induced apoptosis
[0225]Cell death can be divided into apoptosis and necrosis. Among them, cell necrosis will lead to cell membrane rupture, swelling and content leakage, thereby causing a severe inflammatory response, which is mainly controlled by receptor-interacting protein kinase 3 (receptor-interaction kinase 3, RIP3). Under the stimulation of TNF-α, necrosomes containing Rip1 and Rip3 are formed in the cells, and Rip3 in the necrosomes recruits and phosphorylates Mlk1. Phosphorylated Mlk1 translocates to the cell membrane to execute necroptosis, during which phosphorylation of Rip3 is necessary for the recruitment of Mlk1 to necrosis factors, therefore, the phosphorylation process of Rip3 is likely to inhibit cell necrosis and control It is an important target of the inflammatory response caused by it. Studies have shown that protein phosphatase 1B (Ppm1b) can inhibit necroptosis in cultured cells and ...
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