Functional polypeptide and application

A technology with multiple functions and polypeptide fragments, applied in the field of medicine, can solve the problems of neglecting the complexity of AD pathogenesis, and achieve the effect of accelerating the clearance and reducing the content.

Active Publication Date: 2019-06-28
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More than 100 clinical trials were declared to have failed from 1998 to 2017. There are many reasons for the failure of research and development. The well-known neurologist Yan-Jiang Wang also proposed that AD pathology involves multiple

Method used

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  • Functional polypeptide and application
  • Functional polypeptide and application
  • Functional polypeptide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Preparation and purification of the functional polypeptide of the present invention

[0053] In this example, a solid-phase synthesis method was used to synthesize the multifunctional polypeptide MOP. Concrete synthetic steps are as follows:

[0054] Using 9-fluorenylmethoxycarbonyl solid-phase peptide to synthesize said SEQ ID NO:16:Lys-Leu-Val-Phe-Phe-Ala-Ala-Ser-Aib-Leu-Arg-Lys-Leu-Aib-Lys -Arg-Leu-Leu

[0055] (KLVFFA-AS-Aib-LRKL-Aib-KRLL). These procedures were performed using a FocusXC automatic synthesizer (AAPPTEC). The MTT protecting group was removed from the KLVFFAAS-Aib-LRKL-Aib-KRLL sequence base (Rink-KLVFFA-AS-Aib-LRKL-Aib-KRLL-Fmoc), deprotected with TFA / TIS / DCM at 3:5:92 The mixture was shaken for 5 minutes to remove MTT, and the process was repeated twice. Convert the amino group at one end of leucine into carboxylic acid form, add succinic acid and DIEA to DMF to react with the polypeptide, and shake overnight at room temperature. ...

Embodiment 2

[0065] Example 2: The activity of the functional polypeptide of the present invention to inhibit the aggregation of Aβ

[0066] 1. ThT experiment of inhibiting Aβ monomer aggregation

[0067] The Aβ40 of 1mM stock concentration and the functional polypeptide of the present invention (fusion proteins shown in SEQ ID NO:16-22, respectively prepared) were diluted with phosphate buffered saline (PBS; 50mM, pH7.4, containing 150mM KCl), respectively A solution of 100 μM monomeric Aβ40 (mAβ40) and the functional polypeptide of the present invention was obtained. The fresh monomeric solution of Aβ40 (100 μM, 10 μL) was mixed with the functional polypeptide solution of the present invention (100 μM, 0, 1, 5, 10 μL), and the mixture was diluted with PBS to a final concentration of Aβ40 of 20 μM. The inhibitory activity of the functional polypeptide of the present invention on mAβ aggregation was determined in a black 96-well flat bottom plate. Subsequently, the plate was incubated at...

Embodiment 3

[0080] Example 3: MOP inhibits the induced cytotoxicity of Aβ aggregates:

[0081] The well-grown SH-SY5Y single cell suspension was mixed with 5×10 3 The density of cells / well was seeded in a 96-well plate with a volume of 200 μL per well. cells at 37°C, CO 2 After culturing at a concentration of 5% for 24 hours, the original medium was discarded, and the following samples were added to each well:

[0082] The mixture of Aβ40 (200 μM) and MOP incubated for 2 hours (Aβ:MOP=1:1, 1:0.5; 1:0.25), the stop concentration of Aβ40 is 2 μM; The cut-off concentration was 2 [mu]M; the A[beta]40 negative control contained everything except A[beta] and MOP (100% cell viability). The positive control group received 2% SDS solution (0% cell viability).

[0083] After continuing to culture the above 6 groups of cells for 48 hours, add 10 μL of MTT solution (5 mg / mL) to each well, incubate at 37 °C for 3 hours, terminate the culture, add 100 μL DMSO to each well to dissolve the crystals, ...

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Abstract

The invention provides a functional polypeptide. The functional polypeptide includes a polypeptide fragment I identifying and blocking amyloid protein A beta aggregation and a polypeptide fragment IIwhich is a mimic peptide of an apoE receptor binding region, and the polypeptide fragment I and the polypeptide fragment II are linked through a linker peptide or directly linked. The polypeptide caninhibit amyloid protein A beta aggregation, inhibit the of A beta, accelerate removal of A beta through microglia, and the functional polypeptide can be used for preparing drugs for treating the Alzheimer disease.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a functional polypeptide which can be used to prepare medicine for treating Alzheimer's disease. Background technique [0002] Alzheimer's disease (AD) is the most common neurodegenerative disorder and the leading cause of dementia in the elderly population. According to the 2018 World Dementia Report, 50 million people worldwide were living with dementia in 2018 and this number will triple to 152 million by 2050. The total global cost of dementia is estimated to be US$ 1 trillion in 2018 and this figure will increase to US$ 2 trillion by 2030. The pathogenesis of AD is complex, involving abnormal amyloid-β (Aβ) metabolism, hyperphosphorylation of tau protein, oxidative stress, changes in reactive glia and microglia and other pathological events. Among them, the abnormal accumulation and aggregation of Aβ in the brain, known as the "amyloid hypothesis", is considered to be an important ...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/08A61K47/64A61K47/65A61P25/28
Inventor 肖衍宇陈志鹏张珊珊叶俊秀林玲胡子怡
Owner CHINA PHARM UNIV
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