Scorpion toxin polypeptide and method for preparing scorpion toxin polypeptide with similar natural physiological functions
A technology for scorpion toxin and toxin, which is applied in the field of preparing scorpion-derived polypeptide toxin polypeptides with similar natural physiological functions, can solve problems such as decreased drug efficacy, and achieve the effects of short experimental period, high purity and high yield.
Inactive Publication Date: 2017-04-26
合肥科生景肽生物科技有限公司
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Problems solved by technology
However, this method produces a fusion protein instead of the polypeptide itself, and its efficacy is also lower than that of the polypeptide itself (the half-inhibitory concentration IC50 is 13nM)
Method used
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Embodiment 1
[0046] Embodiment 1: 1) the plasmid construction of scorpion toxin polypeptide:
[0047]Obtain the protein sequence information and DNA sequence of the scorpion toxin polypeptide, and carry out gene synthesis, wherein the DNA sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2; use the PCR method to add Factor Xa enzyme digestion before the scorpion toxin The site IEGR was connected to the pET28 vector with His6 affinity tag and SUMO solubilization tag through BamH I and Xho I restriction endonuclease sites to construct pET28-His6-SUMO-Factor Xa-scorpion toxin plasmid. Sequence verification is correct.
[0048] 2) Induced expression of plasmids:
[0049] The plasmid was transformed into Escherichia coli BL21(DE3)gold competent cells, and grown on LB plates containing 50 μg / mL kanamycin in a constant temperature incubator at 37°C for 24 hours. Select a single clone, inoculate it into 4 mL LB tubes containing 50 μg / mL kanamycin, expand and cu...
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The invention provides a scorpion toxin polypeptide and a method for preparing a scorpion toxin polypeptide with similar natural physiological functions. The method comprises the following steps: preparing a gene of the scorpion toxin polypeptide by using a gene synthesis method, establishing the gene into recombinant plasmid with a His6 purification tag and an SUMO hydrotropy tag, and performing high-efficiency expression by using an escherichia coli expression system; after cell crushing, performing nickel column purification, performing enzyme digestion to remove the hydrotropy tag, and performing HPLC (High Performance Liquid Chromatography) purification, thereby finally obtaining the scorpion toxin polypeptide with similar natural physiological functions. According to the method, the preparation process is simple, the cost is low and the yield is high, and the prepared scorpion toxin polypeptide has physiological activities same as those of natural scorpion toxin polypeptides.
Description
[0001] 1. Technical field [0002] The present invention relates to the field of protein and polypeptide drugs, in particular to a method for preparing polypeptide toxin polypeptides similar to natural physiological functions of scorpion-derived polypeptides. [0003] 2. Background technology [0004] There are currently three known ways to obtain scorpion toxin polypeptides or their analogues: one is to extract directly from the crude venom derived from scorpions, and to separate and purify them on a C18 column of reversed-phase high-performance liquid chromatography, from which to separate a variety of single substances in the venom components. This method can obtain natural scorpion toxin polypeptide, but the disadvantage is that the amount of scorpion toxin obtained is very small. After 1 mg of crude poison is separated and purified, only micrograms of target toxin polypeptide can be obtained, and it is inconvenient for large-scale production and operation. The second is t...
Claims
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Login to View More IPC IPC(8): C07K14/435C12N15/12C12N15/70A61P37/02
Inventor 田长麟张隆华肖亮周莉
Owner 合肥科生景肽生物科技有限公司