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Recombinant plasmid of specific ligand of BK channel and recombinant expression method thereof

An expression method and specific technology, applied in the field of recombinant plasmids of specific ligands of BK channels and their recombinant expression fields, can solve the problems of low yield and activity, etc.

Inactive Publication Date: 2015-09-30
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, recombinant Martentoxin (rMartentoxin) is only obtained through conventional in vitro expression methods, and its yield and activity are not high

Method used

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  • Recombinant plasmid of specific ligand of BK channel and recombinant expression method thereof
  • Recombinant plasmid of specific ligand of BK channel and recombinant expression method thereof
  • Recombinant plasmid of specific ligand of BK channel and recombinant expression method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Construction of pGEX-4T-3-Martentoxin plasmid

[0043] Based on the cDNA sequence of MarTX (GenBank No. AF534113.1), two pairs of primers were designed to amplify the coding sequence of MarTX and introduce the restriction site on the vector pGEX-4T-3. MarTX-forward primer (5'-TTC GGATCC TTTGGACTCATAGA-3'), which contains a BamH I restriction site (underlined); MarTX-reverse primer (5'-CTT CCCGGG TTAATCAGTAGCAT-3') contains a Sma I restriction site (underlined). The cDNA sequence of MarTX was inserted between the two restriction sites of BamH I and Sma I of pGEX-4T-3, thereby obtaining the complete expression plasmid pGEX-4T-3-MarTX, such as figure 1 -A shown.

[0044] In this expression system, 6 redundant base pairs appear between the thrombin cleavage site and the cDNA sequence of MarTX. This will lead to two redundant amino acid residues (GS, figure 1 B). Excess base pairs were removed by point mutagenesis as described using the KOD Mutati...

Embodiment 2

[0045] Example 2: In vitro expression and purification of rMarTX

[0046] The expression plasmid pGEX-4T-3-MarTX was transformed into Escherichia coli strain BL21 (DE3), and the strain cells were grown in Luria-Bertani (LB) medium (volume 1 L) containing 0.1 mg / mL ampicillin. The temperature is 37°C. When the bacteria solution OD 600When the value reached 0.4-0.6, Isopropyl-β-d-thiogalactoside (IPTG) was added at a final concentration of 0.5 mM to induce the expression of the toxin protein. E. coli cells were grown continuously at 28 °C for 4 hours. After culturing, cells were collected by centrifugation at 5000 g for 10 min, and resuspended in 70 mL of 1× phosphate buffered saline (PBS, 137 mM NaCl, 4.3 mM Na2HPO4, 2.7 mM KCl, 1.4 mM KH2PO4, pH 7.4). Cells were disrupted in an ice bath and sonicated (4 bursts / min) for 20 min. The lysate was centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was filtered with a 0.45 filter membrane, and passed through an Econo-ch...

example 3

[0050] Example 3: Electrophysiological detection of recombinant rMarTX

[0051] 3.1 Solution preparation for electrophysiological experiments

[0052] In a patch clamp experiment, the extracellular fluid of BK channel (mM): NaCl 135, KCl 5, MgCl 2 1.2, CdCl 2 2.5, HEPES 5, glucose 10 (adjust pH to 7.4 with NaOH); electrode inner solution (mM): NaCl 10, KCl 117, MgSO 4 2, HEPES 10, MgATP 2, EGTA 1 (adjust pH to 7.2 with KOH).

[0053] Extracellular fluid of mKv1.3 and hKv3.1a channels (mM): NaCl 135, KCl 5, MgCl2 1, CaCl2 1.8, HEPES 10, glucose 10 (adjust pH to 7.4 by NaOH); extracellular fluid of hKv4.2 channel (mM): NaCl 125, KCl 2, MgCl2 1, glucose 10, HEPES 10, TEA 20 (adjust pH to 7.4 by NaOH). Electrode inner solution of mKv1.3, hKv3.1a and hKv4.2 (mM): KCl 130, MgCl2 0.5, MgATP 2, EGTA 10, HEPES 10 (adjust pH to 7.3 by KOH).

[0054] 3.2 Transient transfection

[0055] BK channels, mKv1.3, hKv3.1a and hKv4.2 channels were all expressed in HEK293T cells by transi...

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Abstract

The invention relates to a recombinant plasmid of a specific ligand of a BK channel and a recombinant expression method thereof. The recombinant plasmid of the ligand is a base sequence shown in SEQ ID NO:1. The invention provides establishment of an in-vitro expression vector pGEX-4T-3 and technical parameters of a MarTX in-vitro expression method. The short chain scorpion toxin MarTX obtained in the invention is successfully expressed in a pGEX-4T-3 and the molecular weight and bioactivity of the short chain scorpion toxin MarTX are almost the same as those of the natural MarTX; the recombinant MarTX is capable of selectively blocking the BK channel (alpha+beta4) and partially inhibiting the electric current of an mKv1.3 channel but has no obvious modulating effect on hKv4.2 and hKv3.1a.

Description

technical field [0001] The invention relates to a recombinant expression method of a recombinant plasmid of a BK channel specific ligand. Background technique [0002] Among the numerous ion channels, potassium ion channels are the most widely distributed, most subtyped, and most complex ion channels, and exist in almost most organisms. Potassium ion channels control a wide range of biological functions, and play a key role in the generation and propagation of action potentials in excitable cells. Their activation and inactivation characteristics are closely related to the firing mode of action potentials. The content of specific ligands of potassium ion channels in natural products is very small, which is difficult to obtain through traditional separation and purification methods; and due to the limitation of its small molecular weight, it is also difficult to obtain active polypeptides through conventional in vitro expression methods. [0003] At present, although more th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66
Inventor 吉永华施健陶杰祝艳
Owner SHANGHAI UNIV
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