Artificial synthesised scorpion chloride ion neurotoxin gene-rBmK CTa

A chloride ion channel, neurotoxin technology, applied in genetic engineering, plant genetic improvement, medical preparations containing active ingredients, etc.

Inactive Publication Date: 2005-03-23
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the extraction of a single pure protein from crude scorpion venom is not only cumbersome, but also in a small amount, which is far from meeting the needs of scientific research and medical production, especially for those toxins with low content in natural crude scorpion venom. Efficient way of protein

Method used

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  • Artificial synthesised scorpion chloride ion neurotoxin gene-rBmK CTa
  • Artificial synthesised scorpion chloride ion neurotoxin gene-rBmK CTa
  • Artificial synthesised scorpion chloride ion neurotoxin gene-rBmK CTa

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Artificial synthesis of modified genes

[0037] According to the natural BmK CT gene synthesis of 6 oligodeoxyribonucleic acid sequences (see Figure 1C). Among them, Primer 1 and 2 are forward primers, Primer 3 and 4 are reverse primers, and BmK CT (EcoRI) and BmKCT (BamHI) are two-end adapter primers with EcoRI and BamHI restriction sites respectively to connect with pExSecI expression system .

[0038] Primer1 and 3, 2 and 4 were used for PCR, and then the two products were smoothed at the ends. After mixing, the rBmK-CTa gene was obtained by PCR. It was cloned into pGEM-T Easy and sequenced (see Figures 2, 3).

Embodiment 2

[0039] Example 2. Construction of recombinant expression plasmid

[0040] The rBmK-CTa gene was amplified with primers BmK CT (EcoRI) and BmKCT (BamHI), then digested with EcoRI and BamHI, and connected with the expression vector pExSecI cut with the same restriction (see Figure 4).

Embodiment 3

[0041] Example 3. Induced expression and purification of recombinant scorpion chloride channel neurotoxin rBmK CTa

[0042] The recombinant plasmid pExSecI-rBmK CTa was transformed into Escherichia coli BL21(DE3), induced by 0.4mM IPTG at 16°C for 4 hours, SDS-PAGE electrophoresis detected a large amount of the gene expression, accounting for 19.936% of the whole bacterial protein, while the control gene BmK CT The expression level of is only 9.116% (see Figures 5 and 6), indicating that the improved scorpion chloride channel neurotoxin gene rBmK CTa is more than 50% higher than the original gene BmK CT in E. coli.

[0043] After IgG-Sepharose 6 Fast Flow (Pharmacia biotech, Sweden) affinity chromatography, a relatively pure protein was obtained, and 2.4 mg of the protein can be purified from one liter of culture medium (see Figure 7A). Western blot identified the rBmK CTa gene expression product (see Figure 7B). The antibody is the total protein antibody of scorpion venom.

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Abstract

The invention makes 24-site mutation on natural scorpion chlorine ion channel neurotoxin gene BmK CT according to the principle of Escherichia coli partial to codon, designs DNA sequence suitable to be expressed in Escherichia coli BL21 (DE3), adopts PCR technique to complete artificial synthesis of recombinant scorpion chlorine ion channel neurotoxin gene rBmK CTa. On this basis, it clones rBmK CTa into pEXSecI expression system to transfer in the BL21 (DE3), screens and obtains high-performance expressed bacterial strain, detects that the expressed product of the rBmK CTa accounts for 19.936% of the all-bacterium protein by SDS-PAGE electrophoresis, and by affinity chromatography, obtains purer protein with bioactivity, and can obtain 2.4mg protein from one liter culture liquor by purifying. The obtained modified recombinant scorpion chlorine ion channel neurotoxin has inhibition effect on neuroglia cell and can be used in preparing medicines curing diseases by inhibiting neuroglia cell and also be used in research on the space structure and pharmacological activity of scorpion neurotoxin.

Description

Technical field [0001] The invention relates to mutation and genetic engineering, and specifically belongs to DNA recombination technology, and more specifically, is an improved scorpion chloride ion channel neurotoxin gene rBmK CTa and its expression. Background technique: [0002] There are about 15 species of Chinese scorpions, of which Buthus martensii (Buthus martensii) is the most widely distributed and most abundant. In our country, since 1981, some progress has been made in the research of East Asian scorpion. Part of the scorpion toxin has been purified, part of the primary structure has been determined, pharmacological and physiological effects and effects on cell membranes have been studied, and many anti-epilepsy peptides (AEP) with high anti-epilepsy activity have been purified from the scorpion venom (Wang , CG, European Journal of Biochemistry, 2001, 268: 2480). [0003] Glial cells are cells in the human brain that are prone to become cancerous. Glioma cells are k...

Claims

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Application Information

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IPC IPC(8): A61K38/16C07H21/00C07K1/14C12N15/11C12N15/12C12N15/63C12N15/70
Inventor 梁爱华付月君王伟柴宝峰张志云
Owner SHANXI UNIV
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