Fusion polypeptide for inhibiting neurotransmitter secretion and method for delivering it

A technology that combines polypeptides and neurotransmitters, applied in chemical instruments and methods, hybrid peptides, nervous system diseases, etc., can solve the problems that the applicability of the biologically active substances to be delivered has not been clearly confirmed, and achieve Efficiency-promoting and secretion-inhibiting effects

Inactive Publication Date: 2012-11-07
FORHUMANTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effect of these methods on the intracellular delivery of the bioactive substances to be delivered and their general applicability have not yet been definitively demonstrated

Method used

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  • Fusion polypeptide for inhibiting neurotransmitter secretion and method for delivering it
  • Fusion polypeptide for inhibiting neurotransmitter secretion and method for delivering it
  • Fusion polypeptide for inhibiting neurotransmitter secretion and method for delivering it

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: Preparation of PTD-SBD (VBD) conjugate

[0056] (1) Preparation of PTD-SBD (VBD) conjugates

[0057] YARVRRRGPRRGGGEIDTQNRQIDRIMEKAQANKTRIDEANQRATKMLGSG (PTD-GGG-SBD polypeptide)

[0058] According to the method of solid-phase synthesis, a fusion polypeptide (PTD-GGG-VBD polypeptide) whose amino acid sequence is YARVRRRGPRRGGGNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKR was synthesized using a semi-automatic synthesizer (Peti-Syzer Model PSS-510). In order to achieve this purpose, 0.1 mmol of Rink Amide MBHA resin was placed in a standard reactor, and 0.5 mmol of activated Fmoc-G and Fmoc-R (Fmoc-G and Fmoc-R) were added to the reactor. Fmoc-R is the first C-terminal amino acid of the polypeptide to be synthesized), and then the synthesis of the polypeptide is started. In order from C-terminal amino acid to N-terminal amino acid, 0.5 mmol of each corresponding amino acid was condensed 3 times. Using dimethylformamide (DMF) containing 20%...

Embodiment 2

[0061] Example 2: Preparation and purification of expression vectors for fusion polypeptides using microbial expression systems

[0062] (1) Preparation of expression vector

[0063] To construct the base sequence encoding the fusion polypeptide, an N-terminal Hind III cleavage site and a C-terminal BamH I cleavage site were set in pUC19 (purchased from Invitrogen), thereby constructing a template. In order to bind the polyhistidine tag, which enables high expression and easy purification of the fusion polypeptide, to the base sequence encoding the highly expressed protein, the template was isolated using restriction enzymes HindIII and BamH I, and purified using Auiaquick reagent box for purification. The expression vector pPET was treated with restriction enzymes HindIII and BamH I, and purified with Auiaquick purification kit. The purified expression vector is then cloned into the above-prepared base sequence template encoding the fusion polypeptide, thus preparing a re...

Embodiment 3

[0066] Example 3: Effect of Fusion Polypeptide Inhibiting Neurotransmitter Secretion in Test Tubes.

[0067] The effects of the fusion polypeptides prepared in Examples 1 and 2 on inhibiting neurotransmitter secretion were analyzed. To compare the activities, two fragments were prepared separately for SBD and VBD. The fragment samples are as follows:

[0068] Sample 1: SBD (SEQ ID NO: 4)

[0069] Sample 2: VBD (SEQ ID NO: 5)

[0070] Sample 3: Hph-1-GGG-SBD (SEQ ID NO: 14)

[0071] Sample 4: Hph-1-GGG-VBD (SEQ ID NO: 15)

[0072] Sample 5: Tat-GGG-SBD (SEQ ID NO: 16)

[0073] Sample 6: Hph-1-GGG-SBDF1 (SEQ ID NO: 17)

[0074] Sample 7: Hph-1-GGG-SBDF2 (SEQ ID NO: 18)

[0075] Sample 8: Hph-1-GGG-VBDF1 (SEQ ID NO: 19)

[0076] Sample 9: Hph-1-GGG-VBDF2 (SEQ ID NO: 20)

[0077] Hph-1-GGG-SBD (SEQ ID NO: 14)

[0078] YARVRRRGPRRGGGEIDTQNRQIDRIMEKAQANKTRIDEANQRATKMLGSG

[0079] Hph-1-GGG-VBD (SEQ ID. NO.: 15)

[0080] YARVRRRGPRRGGGNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSEL...

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PUM

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Abstract

Disclosed herein is a method of enhancing the efficiency of delivery into cells and local sites by inhibiting the secretion of neurotransmitters using a conjugate of a protein transduction domain (PTD) with domains binding to three kinds of proteins, which form a SNARE complex.

Description

technical field [0001] The present invention relates to inhibiting the secretion of neurotransmitters by using conjugates formed by protein transduction domains (PTDs) and domains capable of binding to three proteins capable of forming SNARE complexes, thereby facilitating their delivery to cells Efficiency in neutralizing local locations. Background technique [0002] Regarding the secretion of neurotransmitters, there are three types of synaptobrevin (vesicle-associated membrane protein, VAMP), SNAP25 (rabbit anti-human synapse-associated membrane protein 25) and syntaxin. The proteins form the SNARE complex, and these three proteins pass through the Ca 2+ Stimulated extracellular secretion; the small synaptic vesicles are present on the surface of synaptic vesicles carrying neurotransmitters, and SNAP25 and syntaxin are present in the presynaptic lipid membrane (see figure 1 ). [0003] Botulinum neurotoxin is often used as an inhibitor of the secretion of neurotransmi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00
CPCC07K19/00A61P17/00A61P25/00A61P25/04A61P29/00A61P43/00A61K38/16
Inventor 李承揆李尚揆
Owner FORHUMANTECH CO LTD
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