Molecular design of targeted potassium channel Kv1.3 active polypeptide and preparation and application thereof

A technology of DNA molecules, hypersensitivity, applied in the field of biology

Active Publication Date: 2014-12-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that it is still challenging to screen and design high-efficiency and low-toxicity (

Method used

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  • Molecular design of targeted potassium channel Kv1.3 active polypeptide and preparation and application thereof
  • Molecular design of targeted potassium channel Kv1.3 active polypeptide and preparation and application thereof
  • Molecular design of targeted potassium channel Kv1.3 active polypeptide and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1: have the acquisition of the DNA molecule of nucleotide sequence as shown in SEQ ID NO:5~7

[0088] 1. Amplification primer design:

[0089] With the nucleotide sequence shown in SEQ ID NO:5 as the template sequence design amplification primer, the primer sequence used for amplifying the DNA molecule of the nucleotide sequence shown in SEQ ID NO:5 is as SEQ ID NO:8 ~11 shown;

[0090] With the nucleotide sequence shown in SEQ ID NO:6 as the template sequence design amplification primers, the primer sequence used for amplifying the DNA molecule of the nucleotide sequence shown in SEQ ID NO:6 is as SEQ ID NO:12 ~15 shown;

[0091] With the nucleotide sequence shown in SEQ ID NO:7 as the template sequence design amplification primers, the primer sequence used for amplifying the DNA molecule of the nucleotide sequence shown in SEQ ID NO:7 is as SEQ ID NO:16 ~19 shown.

[0092] In the process of designing the primers, add the enzyme cleavage sites required f...

Embodiment 2

[0106] Embodiment 2: The transformant construction comprising the nucleotide sequence DNA molecule shown in SEQ ID NO:5~7

[0107] Take the transformant construction process comprising the nucleotide sequence DNA molecule shown in SEQ ID NO: 5 as an example:

[0108] Carry out agarose gel electrophoresis to the PCR amplified product having the nucleotide sequence shown in SEQ ID NO:5, reclaim the target fragment that size is 111bp, obtain having the nucleotide sequence shown in SEQ ID NO:5 DNA molecule.

[0109] EcoR I and Xho I were used to perform double enzyme digestion on the recovered DNA molecule, gel electrophoresis and recovery to obtain a DNA molecule having a cohesive end with the nucleotide sequence shown in SEQ ID NO:5. The double enzyme digestion step was carried out according to the product manual. Using T4 ligase, it was ligated into the expression vector pGEX-6p-1 that had been cut with EcoR I and Xho I to obtain a recombinant vector including the nucleotide ...

Embodiment 3

[0115] Example 3: Expression and purification of polypeptides having an amino acid sequence as shown in SEQ ID NO:2-4

[0116] Take the expression and purification of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 as an example:

[0117] Inoculate the positive bacterial strain identified in Example 2 into 3 mL of LB medium containing 100 μg / mL ampicillin, and after culturing at 37°C for 12 hours, transfer the culture solution to 1 L of fresh LB medium containing 100 μg / mL ampicillin In the medium, after culturing at 37° C. for 4 hours, IPTG was added to make the final concentration 0.1 mM, and the expression was induced at 37° C. for 4 hours.

[0118] Collect the cells in the induced cell solution, suspend them in buffer (50 mM Tris-Cl, 1.0 mM EDTA, pH 8.0), disrupt the cells by ultrasonic, and collect the supernatant by centrifugation. The obtained supernatant was eluted by GST affinity chromatography to obtain a fusion protein solution, and the obtaine...

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Abstract

The invention relates to the field of a biotechnology, especially to a molecular design of a targeted potassium channel Kv1.3 active polypeptide and its preparation and application thereof. The polypeptide provided by the invention has an amino acid sequence as shown in the SEQ ID NO:1. The polypeptide has higher selectivity and lower potential toxic effects than polypeptide which is applied as a potassium channel Kv1.3 blocker in the prior art, and is an efficient low-toxicity blocker of a targeted potassium channel Kv1.3.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the molecular design, preparation and application of a polypeptide targeting potassium ion channel Kv1.3 activity. Background technique [0002] Potassium ion channel Kv1.3 is a protein located on the cell membrane. It is distributed in different tissues such as lymph nodes, vascular endothelium, and spleen, and performs related physiological functions. In recent years, different pathological studies have found that the potassium ion channel Kv1.3 has a significant up-regulation phenomenon, and has become a drug target for the treatment of different diseases. For example, in the T cell-mediated autoimmune disease multiple sclerosis, after the activation of effector memory T cells (effector memory, TEM), the expression number of potassium ion channel Kv1.3 on the cell membrane is up-regulated from about 300 to about 1500 . Similarly, in T cell-mediated autoimmune diseases such as rh...

Claims

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Application Information

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IPC IPC(8): C12N15/63A61P37/00C12N1/19C12N15/11A61K38/17C12N1/21A61P9/00C07K14/47
CPCA61K38/00C07K14/47C12N15/63C12N15/70
Inventor 李文鑫吴英亮曹志贱陈宗运韩松
Owner WUHAN UNIV
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