Method for constructing in-vitro aggregation model of associated protein polyQ of Huntington's disease

A technology of related proteins and aggregation models, applied in the field of genetic engineering, to reduce the cost of screening

Inactive Publication Date: 2011-02-16
SHENZHEN UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0005] The purpose of the present invention is to address the shortcomings of the current artificially synthesized polyQ, to provide a method for establishing...

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  • Method for constructing in-vitro aggregation model of associated protein polyQ of Huntington's disease
  • Method for constructing in-vitro aggregation model of associated protein polyQ of Huntington's disease
  • Method for constructing in-vitro aggregation model of associated protein polyQ of Huntington's disease

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Embodiment 1

[0035] In this embodiment, the length of polyQ is Q42 as an example.

[0036] 1. Experimental materials and methods:

[0037] 1. Experimental materials:

[0038] Escherichia coli strain BL21 Star and plasmid pET28a were preserved by Shenzhen Key Laboratory of Genetic Engineering and purchased from ROCHE Company. Plasmid pEGFP-C1 was purchased from Clontech, USA. GST fusion expression vector pGEX-5X-1 was purchased from Amersham Biosciences. Escherichia coli recombinant plasmid pET28a / EGFP-Q42 was constructed and preserved by our laboratory.

[0039] The construction method of Escherichia coli recombinant plasmid pET28a / EGFP-Q42 is as follows: the plasmid pEGFP-C1 is used as a template, the upstream primer F: 5'-catatggtgagcaagggcgagga-3' and the downstream primer R: 5'-agatctgagtccggacttgt-3' are used as primers, using The EGFP gene fragment was amplified by PCR method. PCR amplification conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, anneal...

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Abstract

The invention relates to a method for constructing an in-vitro aggregation model of associated protein polyQ of Huntington`s disease, which comprises the following steps: constructing an expression vector of fusion protein GST-polyQ(Q36-Q55); making the expression vector express the fusion protein GST-polyQ(Q36-Q55) in expression bacteria; purifying expression products by affinity chromatography to obtain fusion protein GST-polyQ(Q36-Q55); using protease for enzyme cutting to remove the GST tag from the fusion protein GST-polyQ(Q36-Q55) to obtain a polyQ polypeptide; refrigerating and drying the obtained polyQ polypeptide; preparing a polyQ polypeptide solution; carrying out an aggregation reaction on the polyQ polypeptide solution in PBS or Tris-HCl with the pH of 6.5-7.8; and inspecting the aggregation process of the polyQ polypeptide solution by Thioflavin T. The model constructed by the method can be used for screening and dynamics study of active substances preventing Huntington's disease. Compared with artificially synthesized polyQ polypeptide, the method is capable of largely reducing cost for screening active substances.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for establishing an in vitro aggregation model of Huntington's disease-related protein polyQ. Background technique [0002] Huntington's Disease (HD) is an autosomal dominant genetic neurodegenerative disease that mainly occurs in middle-aged and elderly people, and the behavioral ability and cognitive level of patients are impaired. Huntington's disease is caused by HD gene mutation, that is, the abnormal expansion of the CAG repeat sequence encoding polyglutamine in the first exon of HD gene, and the expression product, polyglutamine (polyglutamine) at the amino terminal of huntingtin (Htt) "PolyQ" for short) is abnormally extended, the length of PolyQ exceeds a certain threshold, Q>35 (that is, the number of Q>35), and the expression product (Htt protein) is abnormally aggregated, thereby causing toxicity to neurons and causing disease. Normal Htt protein is ...

Claims

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Application Information

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IPC IPC(8): C12N15/63G01N21/64C07K14/47C07K19/00C07K1/22C12N15/62
Inventor 郑易之李冉辉刘国宝刘昀邹永东
Owner SHENZHEN UNIV
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