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.
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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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