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Recombinant TRPV4 protein and preparation method thereof

A protein and recombinant plasmid technology, which is applied in the field of recombinant TRPV4 protein and preparation, can solve the problems of high cost, low efficiency and application limitation.

Pending Publication Date: 2021-10-01
HUBEI UNIVERSITY OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, related research is to prepare TRPV4 expression vector and transfer it to overexpress in cells, and observe its effect after applying drugs, but the operation cost is high and the efficiency is low
Therefore, the preparation of TRPV4 protein in vitro can greatly reduce the cost of experiments and improve efficiency, but so far, there has been no report on the preparation of TRPV4 protein in vitro, which has limited its application.

Method used

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  • Recombinant TRPV4 protein and preparation method thereof
  • Recombinant TRPV4 protein and preparation method thereof
  • Recombinant TRPV4 protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] This embodiment provides a method for preparing a recombinant GST-TRPV4-6his fusion protein, the specific steps are as follows:

[0045] (1) Design and synthesis of primers

[0046] In the early stage of this study, I purchased an overexpression plasmid containing the entire TRPV4 gene using GV144 as a carrier from Shanghai Jikai Gene Chemical Technology Co., Ltd. After sequencing, the amino acid sequence of the gene was completely consistent with the amino acid sequence of TRPV4 included in NCBI Genebank. Therefore, the cloning of this application will use this plasmid as a template. The GST-TRPV4-6his fusion protein expression plasmid was constructed using pGEX-6p-1 as a vector, and BamHI and XhoI restriction sites, corresponding protective bases and 6his expression genes were added to both ends of the primers respectively. The detailed sequence of the primers is as follows: such as SEQ. Shown in NO.2~SEQ.NO.3.

[0047] (2) Construction and verification of TRPV4 pro...

Embodiment 2

[0055] This embodiment provides a method for preparing a recombinant GST-TRPV4-6his fusion protein. The specific steps are similar to those in Example 1, except that in the protein purification step, the eluate containing 800 mM imidazole diluted to 50 mM imidazole is washed. Concentrate after elution.

Embodiment 3

[0057] This embodiment provides a method for preparing a recombinant GST-TRPV4-6his fusion protein. The specific steps are similar to those in Example 1, except that in the protein purification step, the eluate containing 800 mM imidazole diluted to 200 mM imidazole is washed. Concentrate after elution.

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Abstract

The invention provides a recombinant TRPV4 protein and a preparation method thereof. The recombinant TRPV4 protein contains a TRPV4 protein, a GST tag and a 6his tag. Compared with the prior art, the method has the beneficial effects that the GST tag and the 6his tag are added to the TRPV4 protein at the same time, so that complete TRPV4 protein fragments can be kept in the preparation process of the recombinant TRPV4 protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant TRPV4 protein and a preparation method. Background technique [0002] TPR channel is a family of voltage-independent cation channels, which are widely distributed, can be activated by various stimuli in the internal and external environment of cells, and participate in a variety of important physiological functions. At present, about 30 kinds of TRP channels have been discovered, which can be divided into 7 different subfamilies according to the homology of channel structure: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin transmembrane protein) TRPML (mucolipin), TRPP (polycystin), TRPN (NomPC-like). [0003] In recent years, studies have found that TRPV4, a member of the TRPV subfamily, plays an important regulatory role in various diseases. In the field of traditional Chinese medicine research, many prescription drugs have been found...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12N1/21C12R1/19
CPCC07K14/47C12N15/70C07K2319/23C07K2319/21
Inventor 高小康张晓燕李欧
Owner HUBEI UNIVERSITY OF MEDICINE
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