Cell line capable of stably expressing 6His-Nav1.1 fusion protein and construction

A technology of 6his-nav1.1 and fusion protein, which is applied in the direction of peptides containing His tags, genetically modified cells, animal/human proteins, etc., to achieve the effect of easy purification and enrichment

Active Publication Date: 2019-04-09
CHINA NAT CENT FOR FOOD SAFETY RISK ASSESSMENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the in vitro expression of 6His-Nav1.1 sodium channel fusion protein in human nerve cells

Method used

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  • Cell line capable of stably expressing 6His-Nav1.1 fusion protein and construction
  • Cell line capable of stably expressing 6His-Nav1.1 fusion protein and construction
  • Cell line capable of stably expressing 6His-Nav1.1 fusion protein and construction

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Experimental program
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Effect test

Embodiment 1

[0033] This embodiment provides a 6His-tagged NaV1.1 fusion protein expression cell line and its construction method, comprising the following steps:

[0034] NaV1.1 gene information: human NaV1.1 (SCN1A), NM_001165963, CDS size is 6030bp. As shown in SEQID No.1:

[0035]

[0036] 1. 6His-NaV1.1 plasmid synthesis

[0037] A KpnI restriction site (ggtacc) was added to the 5' end of the CDS of NaV1.1, the stop codon TAA was removed from the 3' end, and a 6His tag + stop codon TAA+XhoI restriction site (catcatcatcatcatcat+taa+ctcgag) was added. His tag is added to the C-terminus of Nav1.1 protein to form the sequence shown in SEQ ID No.2.

[0038] SEQ ID No.2:

[0039] GGTACC CTCGAG

[0040] In SEQ ID No.2, 5' end GGTACC is the KpnI restriction site, For the initial password, 3' end CATCATCATCATCATCAT for the 6His tag, for the termination password, CTCGAG It is the XhoI restriction site. SEQ IDNo.2 removes the KpnI restriction site GGTACC , XhoI restrictio...

Embodiment 2、6

[0055] Expression of embodiment 2, 6His-Nav1.1 fusion protein

[0056] Example 1 After the cell line stably expressing the 6His-Nav1.1 fusion protein is screened out, HEK293 cells can be conventionally cultured and subcultured. which is:

[0057] The medium is: DMEM supplemented with FBS (fetal bovine serum) at a final concentration of 10%, and Hygromycin B (hygromycin B) at a final concentration of 200 μg / ml.

[0058] The culture conditions are: at 37°C, 5% CO 2 Culture in an incubator; the cells need to be subcultured when they grow to about 80%.

[0059] Subculture steps include: culture based on heating in a 37°C water bath until the medium temperature reaches or exceeds room temperature; absorb the original medium in the culture dish, wash it once with PBS, add 0.25% trypsin to digest for about 1min, add medium to stop digestion, Gently pipette the cells off the surface of the culture dish, and at the same time there are no cell clumps visible to the naked eye in the...

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Abstract

The invention provides a cell line capable of stably expressing 6His-Nav1.1 fusion protein and construction. The cell line is prepared by transfecting an HEK293 cell with a recombinant vector carryinga sequence shown in SEQ ID No.3. The cell line can stably express the 6His-Nav1.1 fusion protein with bioactivity, Nav1.1 is easier to purify and enrich by the aid of a 6His label carried by the protein, a biological recognition aglucone and a toxicity research target are provided for Nav1.1 ion channel research, high-throughput screening of new drugs and unknown toxins, and an important biological affinity material is provided for screening, separation and identification of nerve drugs and neurotoxins. Meanwhile, a core raw material is provided for development of biosensors and establishmentof a high-throughput screening technology.

Description

technical field [0001] The invention relates to the technical field of gene recombination and protein expression, in particular to the stable expression cell line and construction of 6His-Nav1.1 fusion protein. Background technique [0002] Voltage-gated sodium channels (Voltage-gated sodium channels, VGSCs) are membrane proteins widely present on excitable cell membranes in the nervous system, heart, muscle and other tissues of animals, and participate in the generation and conduction of action potentials. So far, nine subtypes have been isolated and identified in mammals: Nav1.1~Nav1.9. Among them, Nav1.1 mainly exists in human nerve cells, and is the specific target of many nerve drugs and neurotoxins. After binding, it acts as an antagonist or an agonist, thereby affecting the conduction of action potentials and excitatory signals between nerves and muscles. [0003] As a transmembrane protein, the in vitro expression of VGSCs is extremely difficult. VGSCs are composed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/62C12P21/00C12R1/91
CPCC07K14/705C07K2319/21C12N15/85C12N2510/02
Inventor 周爽赵云峰吴永宁李英骥关立照郑双佳
Owner CHINA NAT CENT FOR FOOD SAFETY RISK ASSESSMENT
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